electron microscopy in cell biology

1

The Use of Electron Microscopyin Cell Biology

Gareth Griffiths•Q1

1 Introduction 41.1 Why Do We Need EM in Cell Biology? 41.1.1 The Strategy 5

2 Specimen Preparation 7

3 Ambient Temperature EM Methods 73.1 Negative Staining of Particles 73.2 Positive-negative Staining Approaches 93.3 Classical Resin Embedding 113.4 Chemical Fixation 123.5 Postfixation 133.6 Dehydration and Embedding 133.7 Methacrylate-based Embedding 143.8 Plastic Sectioning 163.9 The Tokuyasu Sucrose Embedding – Cryosection Method 173.10 On-section Immunogold-labeling Procedure 203.10.1 Step 2 – Antibody Labeling 223.10.2 Step 3 – Gold 223.11 Preembedding Labeling Methods 233.12 Immunohistochemical Approaches using Horseradish

Peroxidase and Cytochemistry 253.12.1 The Use of Microwave Technology for Specimen Preparation 25

4 Cryo-EM Approaches 264.1 Vitrification 264.2 The Bare-grid Method for Particulate Specimens 264.3 Vitrification of Larger Material 274.4 The Cellulose Capillary Tube 27

2 The Use of Electron Microscopy in Cell Biology

4.5 The Fine-needle Biopsy 284.6 Hydrated Cryosectioning 294.7 Freeze-substitution 294.8 Freeze-fracture and Replica Methods 314.9 Simple Shadowing Methods 314.10 The Kleinschmidt and other EM Methods for Nucleic Acids 324.11 EM Autoradiography 33

5 EM-Visualization at Ambient Temperatures 33

6 EM at Cold Temperatures 376.1 EM Tomography 386.2 Low- and High-resolution SEM 406.3 Critical Point Drying 426.4 Freeze-drying and Cryo-SEM 446.5 Coating Techniques for Cryo-SEM 476.6 Scanning Transmission EM 476.7 X ray Elemental Microanalysis by EM 476.8 Energy-filtering Transmission Electron Microscopy (EFTEM) 50

7 Stereology 507.1 Relative Area and Volume 517.2 Relative Profile Length and Surface Area 517.3 Absolute Volume 527.4 Surfaces and Volumes in Practice 527.5 Correlative Light and Electron Microscopy 537.6 Interpretation in EM 547.7 Which Technique for Which Question? 547.8 Final Comment 56

Acknowledgments 56

Bibliography 56

Keywords

Transmission Electron Microscopy (TEM)Electrons passing through the specimen form the image. Transmission of electronsthrough the specimen depends on the accelerating voltage, routinely 80 to 120 KV forspecimens thinner than 300 nm, but for thicker specimens, or for higher resolutionvoltages up to 1000 K can be used.

The Use of Electron Microscopy in Cell Biology 3

EM gridA 3-mm diameter, 0.01-mm (10–15 µm) thick disc of metal (usually copper) withregular perforations. The specimen is placed on the grid and those parts of thespecimen that lie across the perforations are visualized in the EM.

Scanning Electron Microscopy (SEM)This approach is used to visualize the specimen surface. An electron probe scans thesurface and the secondary electrons are recorded in relation to the moving point ofthe probe.

Specimen PreparationThe foundation of all EM. These are highly empirical methods that have beendeveloped for all EM approaches, which allow the specimens to be imaged successfully.For TEM, the specimen must either be particulate, generally thinner than 500 nm, or itmust be sectioned to that thickness, or thinner, after embedding in plastic, or aftercryoimmobilization.

VitrificationThe process whereby liquid water is cooled so rapidly that the water molecules solidifywithout crystallization to form amorphous or vitreous ice. A vitrified specimen isconsidered to be cryoimmobilized.

Freeze SubstitutionVitrified specimens are infiltrated at low temperatures with solvents, usually withheavy metal stains such as osmium tetroxide or uranyl acetate. Following this lowtemperature stabilization, the specimen is infiltrated with resin at low temperaturefollowed by ultraviolet light polymerization below −20 ◦C, or the specimen temperatureis raised to room temperature prior to room temperature resin embedding.

Freeze-Fracture/ReplicaA vitrified specimen is planarly split and coated with a thin layer of metal (such asplatinum, tungsten, or tantalum). The biological material is digested away and thereplica is placed on an EM grid or viewed in the frozen- hydrated state in a SEM. Auseful modification, freeze-etching, involves subliming a surface layer of watermolecules to reveal details of some internal structures before shadowing. For particlesand macromolecular complexes, metal shadowing can be used to form a replicawithout the freezing step. Glycerol spraying is a related technique where the sample issprayed into a glycerol suspension and then rotary-shadowed and viewed by TEM.

Immunogold LabelingVisualization of antigens on sections, or on the surface of isolated particles using(primary) antibodies. These are detected generally using an additional reactioncontaining gold particles that have bound (secondary) antibodies, or protein-A, thatrecognize the primary antibodies.

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TomographyThe procedure for collecting multiple tilt images of a set of particles, or structures in(mostly) thick sections or isolated particles and collating the information computationallyto provide a three-dimensional model of the structure.

Image AnalysisComputing methods used to improve the signal-to-noise ratio of EM images. They arebased on averaging the information from two-dimensional data sets and can reveal thethree-dimensional structure.

StereologyA set of sampling tools and geometrical probability–based principles that allow one toestimate relative and absolute three-dimensional structural quantities (such as volume,surface, length and number), and spatial relationships, from sectional images.

� Most of the organelles discovered after 1945 were first seen by electron microscopy(EM) in thin sections of resin-embedded cells. Since that time, the technicaladvances in EM instrumentation and specimen preparation have been enormous,a consequence of physicists, engineers, and biologists working together. Thesemethods now allow cell biologists to visualize unperturbed cell structures over arange of dimensions, from the atomic resolution in the case of single proteinsor macromolecular complexes to the range from 2 nm and up in the case ofcellular organelles. This level of resolution is far beyond what can be seen by lightmicroscopy. This review will cover the essentials of a range of methods for scientistsinvestigating cell biology using EM. The major emphasis will be on resolution levelspoorer than 1 to 3 nm. In all these methods, the most crucial parameter is specimenpreparation and, without doubt, the ‘‘gold- standard’’ reference technique, whichinvolves vitrification by rapid freezing, in conjunction with cryo-EM.

1Introduction

1.1Why Do We Need EM in Cell Biology?

The main goal of this chapter is to con-vince the reader, especially cell biologists,that electron microscopy (EM) offers theman incredibly powerful set of approachesfor analyzing the ultrastructure of cellsand their components. Moreover, these

methods have been developed to covera vast scale of detail, ranging from theatomic level to the level of whole or-ganisms. While high-resolution cryo-EMis thriving, the use of lower resolutionEM has dropped considerably in recenttimes in cutting-edge cell biology research.There is no doubt that this downwardtrend is a direct effect of the availabilityof increasingly sophisticated state-of-the-art light microscopy (LM) approaches

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that can, for example, detect individualfluorescent molecules under ideal condi-tions. Recently, even the long-consideredlower limit to LM resolution (0.2 µm) hasbeen reduced for detecting fluorescentmolecules to below 0.1 µm. Such devel-opments, while clearly very important,may even deceive more cell biologists intothinking that LM approaches can now doeverything that was earlier considered inthe realm of EM.

There is, however, a fundamental flaw tothis argument. Even if one can see a singlefluorescently labeled molecule in the cell,there is still the fundamental limitationwith LM in relating that signal to the definedstructures in the cell. An EM-thin sectioncan reveal enormous detail of definedstructures, such as membranes, filaments,or ribosomes; when this section is labeledwith immunogold, the gold particles canbe assigned to defined structures with highprecision (within 8 to 20 nm). However, atthe LM level, the fluorescence signal ismostly seen only as a nondescript blob.Often, one compares the immunolabeledsignal from one protein in one colorwith respect to a compartment referencemarker seen in a separate color. However,for such a reference marker to be useful,one needs to have shown first by EM thatit is indeed restricted to the structure ofinterest. Figure 1(a) shows an exampleof immunofluorescence labeling of theGolgi complex, while Fig. 1(b) reveals howmuch more information becomes availablewhen the same specimen is visualizedat the EM level. Both methods provideimportant information, but at different,complementary scales.

State-of-the-art LM is nevertheless, quitejustifiably at the center stage of cell biology.In contrast to EM analysis, which alwayspresents snap-shots of processes, videoanalysis by LM can allow one to see

dynamic events in real time within cells.One of the ‘‘take-home’’ messages of thisreview is to point out that for moderncell biology studies one needs both LM,to see an overview of the ‘‘forest,’’ andEM to see the details of the ‘‘trees.’’ I willtry to demonstrate that state-of-the-art EMmethods should be standard technology inall laboratories involved in molecular cellbiology research. It should also be pointedout that the expertise needed to executethese methods at the highest level is rapidlydwindling, as fewer specialist groups areavailable to keep these methods alive. Ihope this chapter will also contribute toan awareness that this situation can, andmust, be reversed.

1.1.1 The StrategyThe spectrum of EM techniques of interestto cell biologists is quite broad. Foreach method, one can easily write awhole book, and many books are indeedavailable. Several of these methods aretechnically quite demanding. Given thespace limitation and the need to coveralmost all techniques, the approach I havetaken is to explain the basic conceptsbehind each approach so that a totalbeginner could understand how eachtechnique operates and what it is good for.For the most commonly used methods,photographs, and schematic diagrams areprovided to show those details that areimportant for the method to work.

The strategy I have followed is to startwith live cells or organisms, or isolatedbiological material in an aqueous solutionand describe the two fundamentally differ-ent ways of preparing these specimens fortransmission – EM-conventional chemicalmethods, and the cryobased approaches.Some of these conventional methods, aswell as one partly cryoapproach, can beused for the detection of antigens using

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Fig. 1 Comparison of LM and EM.(a) Shows an immunofluorescencemicrograph showing labeling for atrans-Golgi protein sialyl transferase. AHela cell line stably transfected with aconstruct containing sialyl transferasetagged with a domain from the vesicularstomatitis virus G-protein (provided byTommy Nilsson) was fixed with 3%formaldehyde followed by apermeabilization with 0.1% TritonX-100. The cell was then labeled with arabbit antibody against G-protein tagfollowed by a secondary antibodycoupled to rhodamine. The typicalribbon-shaped appearance of the redGolgi complex is evident, next to thenucleus. The latter is revealed usingDAPI staining for DNA that was done atthe end of immunolabeling reaction. (b)Shows a Tokuyasu cryosection of thesame specimen labeled with the sameantibody and protein-A gold(arrowhead). Note the significantamount of labeling on the trans-side ofthe Golgi. There is also sparse label onmembranes of some endocyticstructures (E). EE – represents the earlyendosomes, evident by their sparselabeling for 5 nm gold-BSA (arrow) thatwas internalized for 5 min beforefixation. Bars = (a) 10 µm and(b) 100 nm. Micrographs courtesy ofVeronika Neubrand and AnjaHabermann.

immunogold labeling; the essence of thesemethods will be covered.

At the end of these preparation meth-ods, the specimens are placed on an EMgrid. The two different ways of imagingthese specimens at room temperature orusing a cryostage are then described. Arecently perfected approach for obtainingthree-dimensional models from a seriesof tilted images by tomography will nextbe summarized, followed by two sets ofmethods that visualize (mostly) surfacestructure, namely freeze-fracture (etch-ing)/replica methods for transmission

electron microscopy (TEM) and low- andhigh-resolution scanning EM. The abilityto use scanning transmission EM (STEM)for mass determination and X-ray micro-analysis for the detection of some ions willbe briefly mentioned. The powerful use ofstereology to quantify structural parame-ters from sections will then be outlined.Following a brief description of methodsthat can be used to correlate LM with EMdata, and a note of caution about interpret-ing thin section images, I end by offeringsome guidance on which technique is ap-propriate for which scientific question.

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2Specimen Preparation

Before a specimen can be visualizedby transmission EM, it must fulfil acrucial criterion – it must be very thin (lessthan about 0.4/0.5 µm) for electrons topass through it. In all transmission-EMapproaches, the specimen is mounted on a3-mm diameter metal (usually copper) grid(0.01 mm in thickness). This supports thespecimen between the grid bars and allowselectrons to pass through the specimenthat lies across the holes in the grid.The grid is usually covered with a thinplastic film (such as formvar or collodion)and preferably coated with a thin layerof evaporated carbon before attachingthe specimen. This provides stability byreducing surface charge buildup. Theevaporator is a prominent machine inalmost all EM laboratories.

In practice, there are two different pos-sibilities for adsorbing the specimen ontothe grid. If the specimen is particulate, thatis, a discrete entity that is no larger than,at most 0.5 µm in height, it can be directlyadsorbed onto the grid. Thin regions ofwhole cells grown on EM-grids can alsobe analyzed this way, using a newly de-veloped cryo-EM approach (see Sect. 6.1on Tomography, below). If, however, thestructure is larger or, more commonly, onewants to see structures inside organelles,cells, or tissues, one must embed the speci-men, or solidify it by vitrification, and thencut thin (usually 50–300 nm) sections. Analternative approach is to freeze-fracturethe specimen and analyze the fracturedsurface, or after etching, the subsurfacetopography. Both TEM and scanning elec-tron microscopy (SEM) can be used tovisualize these specimens.

The key to the analysis of any cell struc-ture, at any scale, is the ability to ‘‘fix’’

the structure, either by chemical cross-linking, or, more preferably, by arresting itphysically in its aqueous environment byfreezing. Although chemical fixatives havebeen used successfully for EM since theearly 1940s (see Fig. 17), the introductionof the concept of cooling into the vitrifiedstate has now emerged as the fundamentalinnovation in EM-specimen preparation.The ability to vitrify a specimen withinmilliseconds, in conjunction with a phys-ical proof that the specimen is indeed ina vitreous state, now offers the best pos-sible method that provides a fundamentalframe of reference for all EM-specimenpreparation methods.

Native biological material is extremelyfragile and sensitive to electron beamdamage. For this reason, a series ofmethods have been developed to optimizespecimen preparation, the sine qua nonof all EM. While one kind of approach,environmental SEM (ESEM) is starting toprovide a means of imaging specimens(such as whole cells) in their aqueousenvironment, this is still in its infancy.This approach will not be discussedfurther here.

3Ambient Temperature EM Methods

3.1Negative Staining of Particles

The simplest and most rapid approachin EM is ‘‘classical’’ negative-staining(Figs. 2, 4). This approach is usefulfor proteins, macromolecular complexes,viruses, (small) bacteria, and isolatedorganelle fractions. Particles are adsorbedonto EM-grids having a suitable surface.This surface is crucial for success andin most cases the grids are covered

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Fig. 2 Negative staining in practice. In (a) the grids are seen floating on solution,they can be moved from one drop to the next using a fine forceps. (b) After the finalstaining reaction, the grid is lifted from the drop of stain and a filter paper is used toremove the excess stain; (c) and (d) show a simple trick to cleanly remove the driedgrid from the forceps. A triangular piece of filter paper piece is inserted in the forcepsand is then used to gently push out the grid cleanly onto a suitable support.

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with a layer of evaporated carbon on asuitable plastic film (e.g. formvar). It isoften an advantage to ‘‘glow discharge’’grids, a procedure whereby air (in alight vacuum) is ionized by ∼500 to1500 V, thereby making the grid surfacehydrophilic (methods are also available tomake grids hydrophobic; these are usefulfor some specimens). Grids are floatedon as little as 3 to 5 µL of an aqueous(usually buffered) solution of the specimenfor a few seconds to minutes to adsorbthe material.

The grid plus absorbed particles are thenrinsed rapidly with water and floated on a(1–5%) solution of a heavy metal stain,such as uranyl acetate, sodium phospho-tungstate, or ammonium molybdate. Ifpure water is not desirable, the salts inthe buffer (which will react with the heavymetals) can be removed by many rapidchanges (1 s each) of the grids on purestain. After a few seconds to 1 min on thefinal drop, the grid is removed with forcepsand the excess stain is partially removedwith filter paper (Fig. 2). A relatively thicklayer (∼100–300 nm) of stain needs to drydown around the specimen. The staining isreferred to as ‘‘negative’’ because the stainfills the spaces around the specimen thatare seen outlined in negative contrast. Ex-amples are shown in Fig. 4(a–c). Althoughtechnically trivial, there are a number of‘‘tricks,’’ and in the hands of specialists,this approach can provide a relatively highlevel of resolution/information..

This approach (and the positive stainingdiscussed below) is easily compatible withimmunogold labeling before the dryingstep. One limitation is that only antigensexposed on the surface of particles can belabeled. In some cases, the particles canbe treated with reagents that can ‘‘openup’’ the particle. In one such example, thereducing agent dithiothreitol (DTT) was

used to expose antigens that are normallyburied within vaccinia virus particles (seeFig. 4b).

3.2Positive-negative Staining Approaches

The adsorption staining procedure de-veloped by Tokuyasu to protect thawedcryosections from drying artifacts is a pow-erful alternative method that is easier tocontrol and to image than negative stain-ing, especially for beginners. The methodis shown in practice in Fig. 3 and schemat-ically in Fig. 8(g). In this approach, thestain adsorbs to some parts of the spec-imen (positive staining) and can be seenbecause of the low background contrast.Some negative-staining effects can alsobe seen, depending on the specimen andthe precise conditions used (see the rightvirus particle in Fig. 4b). This is expectedsince heavy metal compounds used fornegative staining also positively stain bio-logical structures.

This method has several advantages overclassical negative staining. (1) The stainadsorbs to selected structures, usually byionic interactions. Therefore, the differ-ences in intensity of staining in differentparts of the structures provide additionalinformation not available by negative stainmethods. (2) The fact that the overall con-centration of heavy metal stain over thegrid is far less than in negative stain-ing means that beam damage, often aserious problem in negative staining, isfar easier to deal with. (3) This meansthat it is also easier to collect largedata sets, as for tomography due to thereduction in beam-induced damage. Im-ages such as the one shown in Fig. 4(b)are currently being analyzed by this ap-proach. Membrane organelles collapse farless than with normal negative staining

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(a)

(d) (e) (f)

(b) (c)

Fig. 3 Methylcellulose embedding in practice shows the basic techniqueinvolved in methylcellulose embedding. (a) The grid, having been previouslyrinsed in distilled water, is placed on a solution of methylcellulose and uranylacetate on ice. (a–c) The grid is then looped out and the excess stain is removedby touching the loop onto a surface of filter paper (d) The grid is allowed toair-dry. Ideally, the interference colors should be gold, purple, or blue at the endof the technique. (e) and (f) show removal of the grid from the loop•.Q2

(4) Finally, by varying the concentration ofthe stain, one has the possibility in theTokuyasu approach to vary the physicalappearance of the particle; for example, lo-cally high concentration of uranyl acetate

over some structures can reveal nega-tively contrasted thin tubules that are oftenmore difficult to visualize by positive-staining methods. An example is givenin Fig. 4(b).

Fig. 4 Examples of negative and positive staining for EM. (a) Shows a classical negative stainingusing phosphotungstic acid of tobacco mosaic virus (TMV). The typical rod shaped appearance of thisvirus is seen. Some subunits of the virus are also seen end-on. It is evident that these are spherical inprofile. (b) Shows vaccinia virus that has been treated with the reducing agent dithiothreitol (DTT).This treatment is known to break disulfide bonds within the virus. The virus was then embeddedusing the metal cellulose/uranyl acetate mixture as shown in Fig. 3. The increased stabilization ofstructures and the partly positive staining by this procedure allows more details of this relatively largevirus to be seen. The arrowheads indicate two round tubular projections that have been ejected fromthe core of the virus by the• DDT treatments. We believe these may facilitate virus entry into cellsQ3during infection. The particle on the right shows a side-view of another particle. In (c), the same viruspreparation can be seen after conventional negative staining using uranyl acetate. Less fine detail isevident after this approach. Nevertheless, the areas where stain enters the particle can be delineatedby the heavy metal stain. The arrowheads indicate apparent openings between the virus and the coreof the particle. Bars = 100 nm. (a) – Courtesy of Heinz Schwarz; (b) and (c) – from the author.

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3.3Classical Resin Embedding

The majority of thin section analysisworldwide is still carried out usingroom-temperature specimen preparationtechniques in which chemical fixatives,

especially glutaraldehyde, are applied.These approaches are structurally accept-able in so far as the structures theydescribe have been observed by a cry-obased approach; this statement is rea-sonably valid for all known organelles. Fornew structures, their acceptance should

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be based on confirmation by a cryobasedapproach, such as freeze-substitution orusing hydrated cryosections.

By far the most widely used approachfor ultrastructural analysis today, as ithas been for the past 50 years, is theepoxy resin method. This is a witch’sbrew of empirically determined steps forconverting fresh tissue, cells, or isolatedorganelles into a thin section. Althoughthe variety of recipes is greater than inmost cookbooks, the general principleis as described below. For analyzingrepeatedly the same cells or tissueswhose ultrastructure is well known, as inpathology, the conventional plastic-sectionapproach is still the method of choice inmost laboratories. Many useful textbookson EM can be found in many establishedEM laboratories around the world (usuallycollecting dust!).

3.4Chemical Fixation

Glutaraldehyde is the chemical fixative ofchoice for preserving structure for conven-tional EM. The introduction of this fixativewas, without doubt a revolutionary inno-vation for EM in cell biology; a numberof organelles, such as microtubules, werefirst seen only after they could be pre-served with this reagent. It is a powerfulcross-linker of amino groups, especially inproteins and amino-lipids. Although thechemistry involved in this process is verycomplex, and complicated more by the factthat the cross-linking coincides with theprocess of cell death, in practice it is verysimple to apply the fixative to any ‘‘open’’cell system; that is, cells that are accessibleto a solution without having a barrier, suchas a cell wall or a cuticle to block access.If cells are growing in culture, the bestresults are usually achieved by adding the

fixative to the growth medium directly, forthe first few minutes, and then switchingto buffered fixative. Any barrier to the solu-tions, such as cell walls, must be dealt withon an individual basis. The best advice is tocheck how others have prepared the cellsof interest. If one wishes to fix a tissuein situ in an animal, the best procedureis usually perfusion, a nontrivial surgicaloperation. It is important to respect theold rule of thumb that, for optimal results,the specimen pieces should be less than1 mm in all dimensions to ensure goodpenetration of reagents.

When immunolabeling, as opposed topurely structural analyses, is the goal, itis often the case that extensive cross-linking with glutaraldehyde will tend tosterically hinder the access of antibodiesto the antigens. A weaker cross-linker,formaldehyde (which, in solution becomesmethylene glycol) is therefore used forthis purpose, either by itself, or in com-binations with (lower concentrations of)glutaraldehyde. For example, in standardprotocols for the Tokuyasu method (see be-low), cells are fixed for 15 to 60 min in 4%formaldehyde and 0.1 to 0.2% glutaralde-hyde followed by an overnight (or longer)fixation in 4% formaldehyde in a suitablebuffer.

Aldehyde cross-linking generally is aprocess that releases protons. This low-ering of the pH within cells is unlikely tobe protected by the buffer since all the rou-tinely used buffers are charged moleculesthat are thought to enter cells very inef-ficiently. The cross-linking reactions alsoconsume oxygen.

Although this approach can providea faithful presentation of ultrastructure,there are many known examples of speci-men preparation–induced artifacts. Thisusually becomes evident when a less-perturbing cryobased method is used.

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A classical example is the bacterial‘‘mesosome’’; earlier considered a distinctorganelle, it is now known to be a fix-ation artifact, from the use of hydratedcryosections. Another striking exampleof aldehyde-induced artifacts in the frogretina revealed by high-pressure freezingand freeze-substitution has already beenwritten about.

3.5Postfixation

For pure structural studies, but generallynot for immunolabeling, the aldehydefixed tissue is ‘‘postfixed’’ in (0.5–1%)osmium tetroxide (OsO4). One of thevery first micrographs using this fixativefor EM is shown in Fig. 16. This heavymetal can cross-link lipids and protein andadds general contrast to many organelles,especially membranes (enhancing thetrilaminar appearance). It is also clear thatartifacts may be induced, the most strikingbeing the proteolysis of structures suchas actin filaments. It is generally acceptedthat, when used at the low temperaturefor freeze-substitution (see below), the useof OsO4 is generally free of artifacts andis a useful addition to enhance contrast.As pointed out below, OsO4 is generallyincompatible with the polymerization ofmany resins used for immunolabeling(such as lowicryl). Concentrations up to1% in the freeze-substitution schedulemay be used without interfering with UVpolymerization of these resins. Osmiumcan also be successfully used with LRwhite (see below) (see Fig. 7). Manyworkers add a second postfixation stepafter the OsO4 using uranyl acetate, whichbinds to negatively charged phosphategroups in phospholipids, is generallyan excellent stabilizer of lipids. Tissuesare often left in aqueous solutions of

this stain for, for example, 1 h; analternative so-called en bloc procedurethat we often use is to leave specimensovernight in a saturated solution ofuranyl acetate in 70% ethanol prior toembedding.

3.6Dehydration and Embedding

The epoxy resins (e.g. Epon, Spurrs,Araldite) are highly hydrophobic, whilemany of the methacrylates have a lowerhydrophobicity, but are still immisciblewith water. For this reason (as in paraffinembedding for histology), the water mustbe replaced (gradually) with an ascendingseries of ethanol or acetone (which tendsto extract more material than ethanol).At the 100% solvent stage, the resin isgradually mixed with the solvent overa period of a few hours until it canbe placed in pure resin. At this stage,the specimen in resin is mounted intoan embedding mold (Fig. 5b) or othersuitable container (e.g. BEEM capsule)and left overnight at room temperature(at which little polymerization occurs) forcomplete infiltration, before being allowedto polymerize for about 12 h at 60 to 70 ◦C.The specimen is now ready to section.The standard epoxy and araldite resins arequite viscous and it is often a problem topenetrate some specimens, such as manyplant and insect tissues. In 1969, ArthurSpurr introduced his concoction, knownas Spurr resin as a low viscosity epoxy resinalternative. This has become the standardresin for many specimens. Alternative lowviscosity–embedding media are now alsocommercially available. For more detailsthere are many classical textbooks availablein EM laboratories. For an example ofan image of an epoxy resin section, seeFig. 6.

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3.7Methacrylate-based Embedding

After the introduction of the completeTokuyasu method in 1978, this approachwas the section method of choice for im-munolabeling. For the next two to threeyears, all alternative embedding proto-cols were far inferior for this purpose.However, in 1980 appeared the first ina series of papers from Eduard Kellen-berger’s laboratory in Basel, in whichan excellent alternative approach becameavailable. After years of painstaking workin collaboration with an industrial partner(Lowi), Carlemalm et al. were successfulin introducing the first of the Lowicrylresins, the more hydrophilic K4M, and themore hydrophobic HM20. Its great poten-tial for immunogold labeling was shownalready in the first publication. The basicapproach is no different to the epoxy proto-col outlined above; it is simply a questionof which solutions, in which order, andfor how long. However, the resins arepolymerized by UV light rather than byheat.

Although these resins can be used atroom temperature, the goal from theoutset was to develop these resins pre-dominantly for use at low temperatures.In the initial publications, an approach

known as progressive lowering of temperature(PLT) was introduced. Here, starting atthe dehydration step, the temperature isgradually lowered to −35 ◦C. The impor-tance of these resins is that they can bepolymerized at this temperature using UVpolymerization (although they can also bepolymerized by heat). This polymerizationusually takes one to three days and of-ten the blocks need additional curing timeat room temperature in direct sunlight(without glass).

In 1985, this group introduced newresins (K11M) that could be polymer-ized down to −60 ◦C and HM23, whichwill polymerize even at −80 ◦C. Thesebecame especially useful in combina-tion with freeze-substitution (see below,and Figs. 13, 15b). The important con-tributions of Bruno Humbel and MartinMuller toward this approach should alsobe mentioned.

In England, Brian Causton, a chemist,in conjunction with the London Resin (LR)Company, developed an alternative set ofresins LR white and LR gold. These resinsare now widely used for EM immunola-beling, especially in laboratories that lackthe equipment for cryosectioning. As men-tioned, LR white is also compatible withosmium tetroxide treatment and recipesexist for providing beautiful preservation

Fig. 5 Plastic sectioning for EM. In (a), a trimmed Epon block is shown in a Leica ultramicrotomebeing sectioned on a Diatome diamond knife. The 2.5-mm knife-edge is indicated by arrows. A ribbonof sections is seen. This ribbon is only slightly longer in length than the diameter of an EM grid.Normally, one would tease this ribbon away from the knife-edge using a fine eyelash. The grid is thenlowered carefully, film side down, onto the ribbon and pressed gently. The grid is ideally rotated onthe surface of the water so that the water can run off smoothly from the surface of the grid. The grid isthen raised and air dried. An alternative method that avoids section wrinkles is to dip the grid into thewater beneath the sections and then carefully raise the grid at about a 45◦ angle to fish the ribbon.Photograph courtesy of Robert Ranner Leica Microsystems GmbH, Vienna. (b) Shows specimensthat have been polymerized in an embedding mold. The specimens are black following osmiumtreatment (arrows). A piece of paper with specimen details can be conveniently coembedded with thespecimen. Courtesy of Anja Habermann and Maj Britt Hansen.

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in conjunction with immunogold label-ing (Fig. 7).

All the methacrylate-based resins aresectioned in the same way as other plastic-embedded blocks (Fig. 5a). For the morehydrophilic ones, such as Lowicryl K4M,the water level in the trough of theknife boat (Fig. 5a) needs to be lowered,

compared to Epon sectioning, to preventthe sections being pulled behind theknife.

All the resins used for EM are po-tentially toxic, as are the fixatives andappropriate caution should be taken whenhandling them (eye protection, gloves,hood, etc.). Particular care should be

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Fig. 6 Example of an epoxy resinsection. This shows a section of a J774macrophage that had internalizedBSA-conjugated gold particles for 1 hfollowed by a chase in medium free ofgold for 1 h. Under this condition, thegold is in late endosomes andlysosomes (Ly). Subsequently, 1-µmlatex beads (B) were added for 1 hfollowed by a further 3-h chase. Goldcan be seen in late endocytic organellesand in the latex bead phagosomes(arrowhead), after a fusion process.Other organelles, Golgi stacks (G),mitochondria (M), and ER are indicated.

taken when working with the Lowicrylresins that are well known to cause skinproblems.

3.8Plastic Sectioning

The specimen embedded in plastic mustnext be trimmed to make a small blockthat can be pyramidal or rectangularat its tip (usually 0.01–0.1 mm, seeFig. 5a). This can be done manually withrazor blades but is much more pre-cisely done using commercially availableblock trimmers that have metal or dia-mond blades.

The trimmed block is next mounted inan ultramicrotome equipped with a glassknife or, more conveniently using a dia-mond knife (Fig. 5a). The combination ofmodern ultramicrotomes and state-of-the-art diamond knives now enables sections

as thin as 20 nm to be easily and repro-ducibly obtained.

As shown in Fig. 5(a), during plastic sec-tioning, the sections are floated on thesurface of (clean) distilled water wherethey can be manipulated with an eyelashand mounted on a carbon and plastic (e.g.formvar) coated grid. In this simple pro-cedure, the grid surface is brought intocontact with the sections, which are ad-sorbed. The sections are routinely stainedby floating the grids on a solution of, firsturanyl acetate (which may not be neces-sary if it was included before embedding),and second with a solution of lead citrate(for a few minutes only) (see Fig. 2a). Afterrinsing with water and air-drying, the gridsare now ready for imaging.

When immunogold labeling is desired,sections of methacrylate-embedded ma-terial can be labeled, as described be-low. In this case, the staining with

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Fig. 7 Example of embedding in LRWhite. (a) and (b) show rat parotidacinar cells embedded in LR White.Pieces of parotid tissue from a rat werefixed in a mixture of 0.5% glutaraldehydeand 4% formaldehyde under microwaveirradiation, followed by postfixation with1% osmium tetroxide containing 1.5%potassium ferrocyanide (reducedosmium) under microwave irradiationand continued for 30 min on ice. Tissueblocks were embedded in LR White resin,and ultrathin sections were obtained.The ultrathin section mounted on anickel grid was pretreated for etching bya saturated aqueous solution of sodiummetaperiodate for 30 s, followed bytreatment in 1% bovine serum albumin.Immunolabeling was carried out usinganti-GF-1. GF-1 is a monoclonal antibodyrecognizing 105-kDa glycoprotein in theGolgi apparatus of serous exocrine cells.Colloidal gold particles (arrow) can berecognized to distribute on thetrans-cisternae of Golgi apparatus (G).The cells in (b) were additionally reactedfor the trans-Golgi enzyme thiaminepyrophosphatase (TPPase) subsequentto the primary fixation. This reactionproduct is seen as an electron-denseprecipitate in one trans-Golgi cisterna(arrowhead in (b)). The sections werelabeled with an antibody GF-1, revealedby immunogold. The gold particles areseen to be restricted to the trans-side ofthe Golgi stack, colocalizing with theTPPase reaction product (arrowheads).The ER is indicated, as are the secretiongranules (SG), the nucleus (N) andmitochondria (M). Micrographs courtesyof Shohei Yamashina.

(a)

(b)

1 µm

1 µm

heavy metals is carried out after theimmunolabeling. An advantage of thisapproach for immunolabeling is thatthe room-temperature cut sections canbe stored dry on grids indefinitely andstill be immunolabeled when desired.These sections can also be powerfultools for use at the immunofluorescencelevel.

3.9The Tokuyasu SucroseEmbedding – Cryosection Method

An elegant and rapid method for vi-sualizing structure and immunolabelingusing cryosections was pioneered byTokuyasu between the late 1960s and 1978.This is now used extensively worldwide.

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In contrast to the hydrated cryosectionmethod (see below), there are two severerestrictions in obtaining cryosections forimmunolabeling, in addition to the obvi-ous need for the cryomicrotome. One isthat immunolabeling needs to be carriedout above 0 ◦C and the other is that ultra-thin cryosections of fresh specimens aredestroyed when thawed. For this reason,the specimen must be chemically fixedprior to sectioning, usually with a light,fixation protocol (see above).

So, the role of the sectioning processin this approach is simply to be able tocut thin sections of fixed cells and tissuethat can subsequently be immunolabeledwhile fully hydrated at ambient temper-ature. While this may be a disadvantagefor high-resolution preservation, it also of-fers a number of practical advantages incomparison to the hydrated cryosection-ing method. First, because the materialis chemically fixed, the membranes arepermeable to high concentrations of su-crose, which is an excellent cryoprotectant.This means that relatively large piecesof specimen (>1 mm3) can by vitrifiedroutinely by simple immersion in liq-uid nitrogen (which is a relatively poorcoolant). Moreover, sucrose (up to 2.3 M)allows the specimen to be more easilyand uniformly thin sectioned in a cry-oultramicrotome at, for example, −100to −120 ◦C. So, although the method in-volves a vitrification step, it cannot beconsidered a bone fide cryo-EM method.Nevertheless, it is mostly referred to asthe cryoimmuno-EM method. The stepsin this method are shown schematically inFig. 8.

The trimmed specimen is cut with aglass knife, or now more commonly thesuperb new generation of diamond knivesdesigned for cryosectioning. The temper-ature is usually in the range of −90 to

−120 ◦C. At these temperatures, waterobviously freezes and a major differencebetween cryo and plastic sectioning is thatcryosections are cut on a dry knife (i.e. nowater trough). The colder the specimen,the harder the block; for cutting sectionsfor LM, the blocks are made softer by rais-ing the temperature from −50 to −80 ◦C.Many workers also raise the temperatureof the block to −80 ◦C for trimming theblock before cutting thin sections at coldertemperatures. Plasticity of the block isalso an important parameter to vary foroptimal sectioning properties, and com-pounds such as polyvinyl pyrrolidine canbe added to the sucrose infusion in orderto facilitate the sectioning process. At alltemperatures, even the best sections areinitially compressed. They are then pickedup using a 1- to 2-mm loop of thin wire thatcontains a drop of 2.3 M sucrose (Fig. 8d).The sucrose drop is rapidly brought intothe cryochamber and, upon contacting thesections and brought to room temperature,it decompresses; a spherical cell profilethat is significantly compressed after sec-tioning can now regain its spherical shape.Often, this method leads to overstretch-ing of the sections, causing structuralartifacts. For this reason, the use of amixture of sucrose and methylcellulosewas introduced, which could greatly im-prove structural preservation (see Figs. 1b,9, 14). Tokuyasu (personal communica-tion) considers the osmotic pressure to bean additional important factor in sectionpickup. To reduce the impact of high os-motic pressure, using a mixture of 1.7sucrose solution with the detergent Tween80, at a final concentration of 0.01% toretrieve cryosections embedded in 2 to2.3 M sucrose was recommended; we havefound this approach to be very useful.Section compression can also be mini-mized by reducing the sectioning angle

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(a)

(c)

(b)

(d)

(e) (f) (g)

Fig. 8 Schematic illustration of the Tokuyasu method. Thesedrawings, made by Paul Webster, illustrate the basic essentialdetails of the Tokuyasu cryosectioning and labeling method. Allsteps are carried out within the chamber of the cryoultramicrotome.Prior to step (a), the specimen will have been fixed, infused withsucrose, and placed on a specimen pin. (a) Shows the procedurefor trimming the block using a trimming device. Glass knives orspecial diamond trimmers can also be used for this purpose. In (b),the specimen is sectioned on a glass or, in this case, a diamondknife, and the sections are manipulated using an eyelash probe (c).In (d), the sections are picked up on a loop containing 2.3 Msucrose or a mixture of sucrose and methylcellulose. The loop isthen touched onto the surface of a grid and the grid is raised andallowed to be floated (section surface down) on a drop of liquid.(e) Shows the procedure for maintaining a moist chamber aroundthe grids. A petri dish is then layered over the grids, and a piece ofmoist filter paper is added on one side of the petri dish; (f) and (g)show again the principle of drying the grid after embedding in amethyl cellulose mixture (see also Fig. 3).

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of the diamond knife. A new oscillatingdiamond knife is being developed that al-ready looks highly promising in reducingthis problem even further.

Once the sections are thawed on thesurface of the drop of sucrose (or themixture with methylcellulose), the loop isbrought to the surface of an EM grid and,upon contact the sections are transferredto the grid surface. From now until thefinal drying step, the fragile sections mustremain hydrated and not be allowed to dry.The universal procedure for immunogoldlabeling can be applied (see below). Itshould be noted that in addition to its usefor immunolabeling, this method is alsoan excellent and rapid (1–2 h) method forpreserving and visualizing structures inthin sections by EM. It is surprising thatthis method has not been more widelyused, for example, in pathology, as a rapiddiagnostic tool.

Either with or without immunolabel-ing, the final step in the preparation isto dry/embed the sections. Since, as men-tioned, the sections are easily destroyedby air-drying, Tokuyasu spent a num-ber of years developing what in the endturned out to be a surprisingly simpleapproach to protect the sections from

collapsing during the drying process. Hiselegant solution was to float the gridswith sections (that were previously float-ing on pure water) on a mixture of heavymetal stain (usually uranyl acetate) anda polymer (usually methylcellulose, thisis more soluble in the cold) for a fewminutes. The grids are then looped outusing a ∼3.5-mm loop, the excess solu-tion is removed by blotting and the gridis air-dried/embedded (Figs. 3, 8). Afterremoving the grids from the loop with aforceps, they can be visualized by EM. Thissimple procedure revolutionized the use ofthawed cryosections for immunolabelingat the EM level.

3.10On-section Immunogold-labelingProcedure

The localization of specific antigens onisolated particles, or on thin sectionscan be performed using immunogold-labeling methods. A theoretical drawbackof these methods is the need to label thespecimen at physiological temperatures,which limits their use for bone fidecryobased approaches. Nevertheless, thesemethods have been very powerful in a

Fig. 9 Example of using the Tokuyasu technique. These images show cryosections ofglutaraldehyde-fixed• CHO cells showing the distribution of Prion protein using an anti-PrP CQ4monoclonal antibody Fab fragment (R1). (a) Gold labeled PrPC (arrowheads) was found to be highlyenriched in the caveolae at the plasma membrane (PM) (not in this picture) and caveolae-containingmembrane structures in the• TGN around the pericentriolar region;• arrows-centrioles.Q5

Q6

(b) Endocytosed 5-nm protein-A gold particles (which bind specifically to PrPC; arrowheads) indicatethe sites of PrPC molecules that had been taken up via caveolae. These results indicate that typicalearly endosomes do not intersect significantly with the endocytic pathway of PrPC-containingcaveolae. In addition to the typical small tubulo-vesicular structures, many largermultivesicular-bodies (late endosomal/lysosomal profiles, L) were loaded with small gold particlesrepresenting endocytosed PrPC, whereas the transferrin receptor (labeled with an antibody and large,10 nm, gold) localized in nearby and distinct structures. As expected, ER, Golgi complex,mitochondria, and nucleus were not labeled, indicating highly specific labeling in these experiments.G: Golgi complex, n: nucleus. Micrographs courtesy of Peter J. Peters.

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wide spectrum of molecular cell biologicalapplications.

The essence of all immunolabelingis the same for all procedures at theLM and EM level. However, the use ofsections (for LM or EM) obviates theneed for a permeabilization step (seebelow). Subsequently, a minimum of threeincubation steps is required, along withrinsing steps. The first step is to blocknonspecific sites. All proteins (some morethan others) have the capacity to adhere

to (most) surfaces and it is generallythought that this is due mostly to ionicand hydrophobic interactions. In practice,one can usually (but not always) blockthese sites on the sections (and grid)surface by simply floating the grid on adrop of a suitable protein solution for afew minutes. Many groups use differentkinds of sera (e.g. goat serum) for thispurpose, in which case one must besure that this does not interfere with theimmunolabeling reaction. We prefer to

100 nm

n

G

I

100 nm

(a)

(b)

Fig. 9

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use a mixture of fish skin gelatin (froman arctic fish, via the Sigma company)and bovine serum albumin. Whateverone uses, it is recommended to dilutethe antibodies and gold reagent in thissolution for the subsequent labeling steps.In practice, the whole labeling procedureis most conveniently carried out on thesurface of a layer of parafilm (see Fig. 2a).

3.10.1 Step 2 – Antibody LabelingThe grid floating on the block solution istransferred to the surface of a 5- to 10-µLdrop of the ‘‘optimal’’ dilution of antibodyin the blocking solution and left for 30to 60 min in a humid chamber on theparafilm surface (see Fig. 2a).

The problems of antibody specificitymust be seriously considered in evaluatingEM (as for LM) immunolabeling. It isimportant to note that apparent proofof specificity by an immunochemicalapproach such as Western blotting does notnecessarily constitute proof of specificityon the surface of a thin section since theconditions facing the antigen are quitedifferent. It is crucial to realize that thecomplexity of possible interactions on thesurface of a section can give rise to subtleand serious artifacts that may fool theobserver. It is also important to realizethat the numbers of antigens availableat the surface of thin sections can bedeceptively low.

It is crucial to determine the opti-mal concentration of antibodies for thebest signal-to-noise ratio. Above this level,background labeling can increase signifi-cantly. The simplest take-home messageto achieve this is to empirically deter-mine the highest concentration of antibody(which, in some cases, may even meanundiluted antibody!) that does not labelstructures assumed to be free of antigens.The foundation of this assumption is the

available cell biological knowledge on thesystem.

Depending on the gold reagent thatwill be subsequently used, one may needto use a secondary antibody step. Forexample, if one uses protein-A gold (whichis the preferred reagent in many groups,including ours), and one starts with amouse antibody (that mostly do not bindprotein-A), then an intermediate step of,for example, rabbit anti-mouse is requiredbefore applying the protein-A gold. Beforeand after such a step, a series of rinses ondrops of, for example, phosphate-bufferedsaline (PBS) is required for 10 to 15 min.Alternatively, one can use a secondaryantibody conjugated to gold (see below).

3.10.2 Step 3 – GoldAfter the rinses following the last anti-body step, the grids are floated on theempirically determined concentration ofprotein-A gold or an IgG gold conjugate.These conjugates can be easily made inthe laboratory or, they can be obtainedfrom many commercial suppliers. In theabsence of prior information, the optimalconcentration of the gold reagent is thehighest concentration that gives negligi-ble labeling in the absence of a primaryantibody on the sections that have beenexposed to blocking solutions.

Colloidal gold particles consist of puregold, made by reducing gold chloride(HAuCl.2H2O). A number of methodsfor preparing these colloids have beenaround since the days of Michael Faradaybut the method of choice now is thatintroduced by Slot and Geuze. By asimple procedure involving citrate andtannic acid this method produces uniform,spherical particles of any size betweenabout 3 and 17 nm; up to three differentsized particles can be routinely usedfor triple labeling studies. Many proteins

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can be adsorbed fairly stably (but mostlynoncovalently onto the surface of goldparticles), without loss of activity; the mostuseful (in our experience) is protein-A. Thenoncovalent interactions of proteins andother compounds with gold is complexand highly unpredictable; one must beaware of the possibility that any compoundbound to gold may elute from theparticle surface with increasing storagetime. Procedures for single and doublelabeling have been described. Followingthe gold incubation, the sections mustbe rinsed extensively (∼15–20 min) andall salts must be washed away with(preferably double or triple) glass-distilledwater prior to final contrasting with uranyland/or lead salts. The basic proceduresfor plastic and cryosections are describedabove. It should also be noted that anyparticulate material that can be adsorbedto grids can be labeled, but on their outer,solvent accessible surfaces. Only if thestructures can be ‘‘opened up’’ in someway can one potentially access internallylocalized antigens.

3.11Preembedding Labeling Methods

Whereas most EM labeling protocols thesedays is done using thawed cryosectionsor methacrylate sections, there is analternative set of methods referred to aspreembedding labeling that may be preferredfor some applications. The principle hereis to start with whole cells or tissue slices(e.g. 20–50 µm thick cryostat or vibratomeor tissue/chopper fresh sections) andthen treat the material with a solvent ordetergent (or even by freeze-thawing) thatremoves parts of membranes in order toallow antibodies to diffuse into the cellinteriors. One simple method that weoften use is to put the live cells into

distilled water and observe them by lightmicroscopy. When the cells burst (usuallyafter about 10 sfor cultured mammaliancells), fixative is added. In this approach, asin all preembedding methods one acceptsstructural damage at the outset.

One attraction of the preembedding ap-proach is that at the end of the labelingprocedure, the cells are embedded in con-ventional epoxy resins, that is, technologyavailable in every EM lab for cell biology.Since the ultimate goal is to see the sitesof antigens in the context of cell ultrastruc-ture, it is essential to chemically prefixthe material before labeling. However, thisraises a practical dilemma; if cross-linkingis too effective, the antibodies fail to reachmany antigens. If, on the other hand, onecross-links too little, cellular ultrastruc-ture is compromised. Since one needsto sequentially introduce antibodies andsecondary gold reagents, preembeddinglabeling is a highly empirical and un-predictable method. Nevertheless, whenit works, this approach can provide signif-icant information (see Fig. 10).

The essential steps in a typical preem-bedding protocol are as follows:

1. The cells/tissue slices are fixed lightly,usually with 2 to 4% formaldehyde(often mixed with a low concentrationof glutaraldehyde), usually for lessthan 30 min.

2. For labeling intracellular antigens, thecells are permeabilized, most often with0.1 to 0.3% of the detergent Triton X-100 for a few minutes. If the goal isto label antigens on the outer surfaceof cells this step can be left out. Thisstep, in conjunction with step 1, isdifficult to control and is prone toinduce artifacts.

3. The permeabilized cells are treated witha protein blocking solution (see label-ing of sections above) and then with the

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5 µm

Fig. 10 Preembedding labeling. The replication of chromatin insynchronized HeLa cells has been studied using both light and EMimmunodetection.• The inset shows individual small fluorescence fociQ7

corresponding to DNA replication sites during early S-phase (100 min afterrelease of cells from a double thymidine block of DNA replication).Biotin-16-deoxy-UTP was used as a marker of the newly synthesized DNA.This was delivered into cells by means of hypotonic shift procedure and wasvisualized by a secondary, fluorescent antibody against biotin. Thecorresponding EM image is seen in the main figure. For this, the mouseantibiotin was recognized by a 1-nm gold anti-mouse antibody that wassubsequently silver-enhanced. The arrow indicates the sites at the peripheryof the nucleus (N) where the silver-enhanced gold accumulate, whichreflects the DNA replication sites corresponding to the immunofluorescencefoci seen in the inset. Micrographs courtesy of Ivan Raska.

optimally determined concentration ofantibody (in blocking solution) for peri-ods ranging from 30 min to overnight.The excess unbound antibody mustbe removed by multiple rinsing stepswith buffer (e.g. phosphate-bufferedsaline, PBS).

4. The cells are incubated with a goldreagent (usually for 30 min to 2 h atroom temp) that recognizes the an-tibody (as for section labeling); alsohere it is crucial to have the op-timally determined concentration ofmarker. The excess is again rinsedaway.

5. The cells can now be fixed with ahigh concentration of glutaraldehyde

(0.5–2% for 30–60 min) in order toprotect the structure against the sub-sequent steps of dehydration and em-bedding. The remaining procedure isidentical to the protocol given above forepoxy resin embedding.

Accessibility of reagents to antigens isoften a severe problem in preembeddinglabeling, with the difficulties increasingas one looks for antigens deeper in thecell, especially within the nucleus. Bythis approach, a negative result is almostimpossible to evaluate; one can neverbe certain whether or not the reagentshad access to the antigens in a definedstructure. At the primary antibody level,

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antibody fragments such as Fab (whichhave a significant lower avidity than wholeIgG) may improve access. At the goldstep, it makes sense to use the smallestsizes of gold. An important innovationhere was the commercial availability of1 to 2 nm gold particles bound withsecondary antibodies or protein-A. Anumber of studies have shown that thesepenetrate significantly better than 5-nmgold particles. The 1- to 2-nm gold particlesare very difficult to see in a typicalsection. To overcome this problem, metalamplification procedures using silver orgold have long been available and widelyused to enhance the size of the goldparticles. It should be noted that osmiumtetroxide can strip the silver off thegold particles.

At the end of this procedure, thin plasticsections of cells are examined in whichgold particles provide an indication of thesites of antigens.

3.12Immunohistochemical Approaches usingHorseradish Peroxidase and Cytochemistry

For preembedding labeling, it is also possi-ble to visualize the bound antibody using asecondary antibody bound to horseradishperoxidase (HRP). This reagent penetratesinto cells better than gold particles and hasthe advantage that the enzymatic prop-erties of HRP can be used to oxidizediaminobenzidine (DAB) into an insolu-ble polymer that binds O5O4 very well.This cytochemical electron-dense reactionproduct can be used as an indicator of thesite of the antigen. Because of the abil-ity of the reaction product to diffuse, thismethod is rarely successful for cytoplas-mic or nuclear antigens. In the past, it waswidely used to localize antigens within thelumen of membrane organelles. However,

for a number of reasons, this approachcannot be considered a ‘‘state-of-the-art’’method for immunolabeling.

HRP has also been widely used as amarker of endocytic organelles, and cellswill conveniently take up this compoundby endocytosis. More recently, cDNAsencoding for this protein as chimeraswith different targeting signals have beenused to direct the protein to differentbiosynthetic compartments, such as theendoplasmic reticulum and the Golgicomplex. This approach can be very usefulto facilitate structural details of theseorganelles and is likely to be increasinglyimportant as an additional marker fortomographic studies. It has also been usedin an interesting method for visualizingendocytic organelles in whole mount cellpreparations.

For structural identification of mem-brane compartments – an old approach –enzyme cytochemistry is still a use-ful method, although its use has beendeclining.• It is expected that this approach Q10

will be used more often for tomographicstudies. An example is provided in Fig. 7.

3.12.1 The Use of Microwave Technologyfor Specimen PreparationIn 1970, Mayers first introduced the idea ofusing microwaves to enhance the rate andefficiency of fixative cross-linking. Sincethat time, this approach has become widelyused, not only for the fixation step butalso for other steps in specimen prepa-ration. Although the precise effects arestill not fully understood, specimen ex-posure to microwaves results in greatlyenhanced rates of penetration of chem-icals into tissues as well as chemicalreaction rates. In the microwave proces-sor, reactions that take hours to occur bydiffusion alone can occur in seconds to afew minutes. It is important to note that

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standard kitchen microwave ovens are notrecommended, since they are difficult tocontrol and, even with short pulses, tend tooverheat the specimen. Specially designedlaboratory-grade microwave processors arenow available that offer variable, control-lable wattage and specimen temperatures.Temperature control is achieved in part bycirculating large volumes of water throughthe microwave chamber to remove excessheat. In our group, following the lead ofPaul Webster, we now routinely carry outall steps of epoxy resin embedding us-ing this technology. Whereas the overallprocess used to take two to three days,it can now be completed in a few hours.The microwave oven is also likely to bean important tool in the future for im-munocytochemistry by speeding up thepenetration and binding rates of antibodiesfor immunolabeling. Its role in improvingantigen accessibility to antibodies is alsobeing exploited., For an example of tis-sue sections following microwave-assistedfixation, see Fig. 7. It is likely that this tech-nology will play an increasingly importantrole in many aspects of EM in the future.

4Cryo-EM Approaches

4.1Vitrification

The pioneering ideas of Dubochet on theconcept of vitrification was the beginningof a new era in EM. The fact that onerequires such a thin specimen for TEMallowed Dubochet and McDowell to vitrifya thin layer of water suspended over theholes on an EM grid. Electron diffractionconfirmed that the solidified water was notcrystalline but remained amorphous. Thisbreakthrough was the culmination of a

whole era of studies that were initiated byFernandez-Moren in the 1950s.

It is estimated that a freezing rate of105 to 106 ◦C s−1 is required to vitrifywater at ambient pressures. No methodis known that allows cells (or parts ofcells) greater than 10 µm in thickness tobe vitrified under these conditions. Onlyat high pressures can this be achieved toa depth of a few hundreds of microns(see below). Owing to the high rates ofcooling that are used, it has been es-timated that the process of vitrificationarrests the in vivo state within 100 m s−1

of the final perturbation. For this rea-son, it has evocatively been described asthe ‘‘solidified in vivo state.’’ There arefive different cryo-EM methods we shalldiscuss: (1) the bare-grid method for iso-lated particles; (2) hydrated cryosections;(3) freeze-substitution; (4) freeze-fractureand (5) cryo-SEM approaches.

4.2The Bare-grid Method for ParticulateSpecimens

Viruses, small bacteria, isolated organelles,filaments, macromolecular complexes,and the like can easily be vitrified. Theremarkable consequence of the thinnessof these specimens is that an almost triv-ial procedure allows preparations to beroutinely vitrified. Following the innova-tion to use a perforated film grid support,the method involves suspending the spec-imen on such a grid mounted in a simpleguillotine device. For the best results, theatmosphere around the specimen shouldbe kept humid and specialized devices arenow available for this purpose. After abrief blotting on one or both surfaces ofthe specimen, the grid, attached to a for-ceps, is rapidly shot into a ∼2-cm deepchamber of liquid ethane cooled by an

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external container of liquid nitrogen. Allthe subsequent operations such as trans-porting, storage, mounting in cryoholdersand imaging in the electron microscope,are done at liquid nitrogen temperature toavoid devitrification, which occurs above−135 ◦C. The imaging of cryospecimensrequires special care because highly en-ergetic electrons, while passing througha sensitive specimen, can easily destroythe fine structure. The so-called ‘‘low doseimaging’’ has, therefore, been developed(see below). It should be noted that thecryo-EM approach offers the only possi-bility in EM for visualizing details withinthe interior of a structure directly. State-of-the-art examples of images obtained by thebare-grid approach are shown in Figs. 11and 18(a).

This approach has been used for aseries of elegant time-resolved analyses,especially by Nigel Unwin and colleagues.For example, the acetylcholine receptorhas been visualized in different functionalstates. The bare-grid approach can also becombined with immunogold labeling andwith positive staining using heavy metalsalts. This is probably the EM methodwhose usage is increasing the most inlaboratories worldwide.

4.3Vitrification of Larger Material

The cryopreservation of material largerthan 1 µm is a topic that has been exten-sively investigated. Despite earlier com-plications, it is now generally acceptedthat the goal is straightforward; the speci-men must be vitrified if it is to providea faithful reflection of the structure ofthe in vivo state. The hydrated cryosec-tion method (see below) offers the onlyapproach that can allow sections of nativematerial to be evaluated. It is also the only

method for unambiguously confirming,via electron diffraction, that the vitreousstate has indeed been achieved; althoughlarge hexagonal ice crystals can be seendirectly without diffraction, the presenceof the intermediate cubic ice crystals (upto ∼300 nm) cannot. These can also dam-age specimens.

There are different approaches for vitri-fying a layer of biological material thickerthan 1 µm; one can consider the moretraditional methods such as (1) plungingin ethane or propane; (2) jet freezing; (3)slam-freezing on a cooled silver or coppersurface. There is now a consensus that allof these approaches unfortunately, fail inpractice to give a layer of vitrification thatis more than 5 to 10 µm.

A relatively old method, high-pressurefreezing, first introduced by Moor, hasrecently emerged as the technique for vitri-fying substantial pieces of biological tissue.A reasonable consensus in the field acceptsthat a layer up to 200 µm (perhaps thicker)can be vitrified by this approach. High-pressure freezers are now available fromthree commercial companies and the ap-plication of this approach is growing slowlybut steadily, worldwide in established EMlaboratories. It should be emphasized thatthe technology involved is not trivial andthere are many ‘‘technical tricks’’ that canbe learned only from the specialists (this isgenerally true of all EM methods). Exam-ples are shown in• Figs. 13–15(b). Below, Q11

I would like to point out two very power-ful adaptations that, in combination withhigh-pressure freezing, can be very power-ful tools.

4.4The Cellulose Capillary Tube

Hohenberg has introduced the use of cel-lulose microcapillary tubes in order to

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prepare material for• HPF. By capillaryQ12

action, an aqueous suspension can bedrawn into these tubes of 200-µm diam-eter, which can be easily sealed by force.The cellulose wall allows molecules up to∼10 KDa to diffuse freely through it. Cul-tured cells can be grown within the tubeson their preferred media prior to freezing(see Fig. 13(a)). For high-pressure freez-ing, small fragments of these tubes canbe conveniently positioned in the speci-men holder of the machine. This approach

can also be useful with other methods, forexample, epoxy resin embedding or theTokuyasu cryosection method.

4.5The Fine-needle Biopsy

A second innovation by Hohenberg is po-tentially of enormous interest to patholo-gists. A specially designed jet micro-needlesystem has been developed that can cut200-µm thick slices of any living tissue.

(a)

(b) Kinesin decoration

Fig. 11

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These slices are ideal for high-pressurefreezing. The use of this system for tak-ing micro-biopsy specimens from humanpatients is now a routine (and for the pa-tient apparently painless) procedure by thisgroup and their medical collaborators.

4.6Hydrated Cryosectioning

Vitrified thick specimens must be sec-tioned before analysis by TEM. The sim-plest method in theory, that is by farthe most difficult to apply, is the hy-drated cryosection method. The theoryis indeed simple, one vitrifies a piece oftissue and prepares cryosections (at a tem-perature below the recrystallization tem-perature of vitreous water (<−135 ◦C)).These sections can be transferred ontoEM-grids kept under liquid nitrogen untilthey are directly visualized (in the absenceof any chemical) at ∼−160 ◦C, or below. Inprinciple, this method allows direct visual-ization of native structure in the absenceof any chemical.

In practice, the technique is quitedemanding and is hampered by sectioning

problems (cutting artifacts). Foremostamong these is the severe compression(routinely 30–50%) that in fact accompa-nies all sectioning methods (in all othersectioning approaches for EM, the sectionshave the opportunity to stretch (decom-press) on aqueous solutions). It seems,however, that in recent years all the majortechnical problems have found acceptablesolutions and the method is now at thestage where it is ready to fulfil its promises.Moreover, new innovations are on thehorizon, such as the vibrating diamondknife already mentioned that can con-siderably reduce the compression artifactand the use of tomography for providingthree-dimensional information from suchspecimens. For recent examples of thisapproach, see Fig. 12.

4.7Freeze-substitution

While the frozen-hydrated cryosection ap-proach is currently something for a spe-cialist, the approach of freeze-substitutionoffers a much easier-to-apply alternativemethod for visualizing the (close to) in

Fig. 11 Cryo-EM. (a) Cryoelectron micrograph of undecorated microtubules, embedded in vitrousice. When assembled in vitro, microtubules may be formed from different numbers of protofilaments(which can be clearly seen in Fig. 22 after metal shadowing). A computer-assisted 3-D reconstructionin the inset (color) has been carried out on a 15-protofilament microtubule. One of them is markedwith an arrow. The number of protofilaments gives rise to different moiree patterns, which can beseen on a careful inspection of the different tubes. For example, 13-protofilament microtubulesexhibit very straight lines along the axis, while 15-protofilament microtubules show a characteristicalternating pattern of fuzzy and striated regions along the axis. This pattern allows one to identifydifferent types of microtubules. (b) Cryomicrograph of microtubules decorated with kinesin motordomains; the corresponding model is shown in color. The motor domains appear as little globularblobs along the microtubule (arrows). These motor domains also have an intrinsic stabilization effectthat creates long isolated protofilaments. The inset shows a helical 3-D reconstruction of amicrotubule decorated with dimeric ncd domains. Ncd is a kinesin family member that exhibitsretrograde directionality, and a unique microtubule-binding pattern, but otherwise shows strongstructural similarities to conventional kinesin. It is important to realize that cryo-EM is the onlyapproach that provides information about structural features within an object. Images courtesy ofAndy Hoenger.

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(b)

(c)

(d) (e)(a)50 nm

20 nm

Fig. 12 Hydrated cryosections. In this approach,no chemical treatment whatsoever is used. (a)Shows a hydrated cryosection of stallion spermchromatin after partial decondensation with10 mM of the reducing agent DTT. Theindividual DNA filaments, whose diameter is2.7 nm, can be clearly seen, as can theirorganization into a hexagonally arranged liquid

crystal; (c) and (d) show different orientations ofDNA; a diffractogram is evident in (b) showingthe hexagonal spacing of the DNA. (e) Shows ahydrated cryosection through purifiednucleosome cores that have self-organized invitro into precisely aligned columns of particles.Both micrographs courtesy of Jacques Dubochet.

vivo structure. Although in this methodthe specimen is dehydrated and embed-ded in plastic, a comparison of the datafrom hydrated cryosections and freeze-substitution has nevertheless convincedmost specialists that the latter approachoffers many advantages over the for-mer, except at the highest levels ofresolution.

Freeze-substitution is an old EM methodthat went out of favor only to emergeover the past decade as the best rou-tine method for preparing sections offaithfully preserved cellular material,thanks especially to the pioneering workby the groups of Muller and Stein-brecht. It can provide not only rela-tively high quality structural preserva-tion but also the opportunity to la-bel the sections (in contrast to hy-drated cryosections) using immunogoldmethods.

The fact that high-pressure freezing, but noother approach, allows vitrification of up to

hundreds of µm of depth in the specimennow makes this approach the method ofchoice for freeze-substitution.

The principle behind freeze-substitutionis to infiltrate the vitrified specimen withsolvents at low temperature that, over aperiod of many hours to days, graduallyreplace the water with the solvent (oftenmixed with stabilizing ‘‘fixatives,’’ suchas OsO4 (that cross-link lipids as thetemperature is raised), or uranyl acetate(that can stabilize the head groups ofphospholipids). The approach can alsobe carried out without any chemicalfixatives. The idea here is to start theprocess of switching the specimen froma water-containing (vitreous) medium to asolvent that is compatible with the plasticembedding media at low temperatures(see below) while the structure of thebiomolecules is still rigid. This is routinelydone at, or colder than −80 ◦C. It cannotbe ruled out that some cubic ice crystalsmay form under these conditions but even

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if they do they are not noticeable at thelevels of resolution usually desired.

In freeze-substitution, the tissue is eitherchemically fixed at low temperature andleft to fix as the specimen is slowlywarmed up, or left unfixed and embeddedin specially developed methacrylate resins(such as Lowicryl HM23) at −70 to −90 ◦C,using UV polymerization. When labelingof antigens is not required, the specimen iswarmed to room temperature, embeddedin Epoxy resin, and then sectioned as inthe conventional method. Figures 13 and15 show images made using this approach.An alternative method that is not oftenused but may be appropriate for somespecimens is to combine freeze-dryingwith freeze-substitution.

4.8Freeze-fracture and Replica Methods

In the 1960s and early 1970s, freeze-fracture was a widely used approach,especially for the study of membranes.Although now restricted to a few specialistgroups in the world, it is still the onlymethod that can allow the investigatorto see the insides of membranes. Smallpieces of specimen are first vitrified, usinga method such as slam-freezing againsta cooled copper block or high-pressurefreezing. Subsequently, the specimen isfractured, either with a knife, or byphysically separating the frozen specimenthat is sandwiched between two metalplates. The freshly opened surface iseither evaporated with a metal replicadirectly, to reveal the surface details ofthe cut specimen, or the specimen is firstallowed to warm up briefly to ∼−90 ◦Cin order to etch away a layer of water.In this case, the replica also reveals somesubsurface details; this is especially usefulfor visualizing details of the cytoskeleton,

as evident in the pioneering studies of JohnHeuser, Tom Reese, Nobutaka Hirokawaand others. The underlying tissue isremoved from the metal replica by harshconditions such as immersion in chromicacid and the fine replica is attached toan EM grid and visualized by ambienttemperature TEM. Thus, although this isa bona fide cryobased method, the replicaof the frozen surface is finally visualized atroom temperature.

A number of methods have been devel-oped for combining freeze-fracture withimmunolabeling. An especially interestingapproach has already been developed; thismethod uses the detergent •SDS to par- Q13

tially digest biological material from thereplica while retaining integral membraneproteins whose exposed cytoplasmic do-main are accessible for immunolabeling.Figure 16 shows two striking examples us-ing this approach.

4.9Simple Shadowing Methods

Since the dawn of the EM era, simple metalshadowing methods have proven useful asa substitute for negative staining that showparticle surface structure. Any isolated par-ticles, such as those suitable for the bare-grid cryo-EM negative-staining approachescan be usefully examined using this ap-proach. When done well, the appearanceof the structure can appear more three-dimensional than with negative staining.In the simplest instances robust struc-tures can be air-dried before evaporatingmetal in a unidirectional, or rotary fashion.Better results are usually obtained after amore gentle preparation method, such asfreeze-drying. Two examples are shown inFig. 23. Another related method is glycerolspraying whereby the sample in glycerol issprayed, then rotary shadowed and viewed

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by TEM. This approach has been widelyused to visualize isolated nucleic acids andattached complexes.

4.10The Kleinschmidt and other EM Methodsfor Nucleic Acids

A useful method for visualizing DNA orRNA molecules, and attached complexes

was developed by Kleinschmidt in 1959.The molecules are adsorbed to a thin, pos-itively charged protein monolayer spreadon a water surface. Cytochrome-C is themost commonly used spreading agent.The film is then picked up on a speci-men grid, contrasted with stain (e.g. uranylacetate) or by rotary shadowing with plat-inum, and observed in the microscope.This method may not have the resolution

(a)

1 µm

Tube wall

I

G

1 µm

(b)

PM

M

N

Fig. 13

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of the cryo-EM method but is more suitablefor estimating parameters such as thelength of the fibers.

About 10 years after the introduction ofthe Kleinschmidt method, the heterodu-plex analysis for mapping regions of basesequence homology in nucleic acids wasestablished, particularly in combinationwith formamide as a denaturating agent.The use of a detergent film (BAC) to re-place cytochrome-C later allowed a betterresolution of the nucleic acid strands.

4.11EM Autoradiography

Until the 1970s, the use of EM autora-diography had a central place in cellbiological research. In this approach, aradioactive molecule is introduced into thecells/tissues as a ‘‘pulse’’ of signal; the clas-sical example was the use of radioactiveamino acids or sugars that are incorpo-rated into newly synthesized proteins orglycoproteins. By removing the radioactiveprecursor, a period of ‘‘chase’’ allows thetracer to be followed sequentially in thecell. This approach was used to follow theER to the Golgi pathway for secretory pro-teins and glycoproteins by the groups ofPalade and Leblond in the 1960s. At theend of the experiment, the cells/tissues arechemically fixed and embedded in epoxy

resins and sectioned. Subsequently, a thinfilm of radioactivity- (and light-) sensitivephotographic emulsion is layered on thesection surface under a red light source inthe dark room. The sections must then beincubated for days, weeks, or even monthsfor enough radioactive encounters to in-teract with the film. At the end of theincubation, the film is developed and visu-alized under the EM.

Although in principle an excellentmethod to follow defined molecules, a sig-nificant disadvantage is the relatively lowresolution, at best 0.5 µm. This, plus thelong waiting period, has resulted in itsalmost total disappearance from the cellbiological scene during the past 20 years.Cell biological problems that used to be an-alyzed by autoradiography are now betterresolved by use of specific antibodies; ki-netic results can then be achieved by use ofspecific inhibitors, such as cycloheximide,that can help to synchronize the synthe-sis of proteins, for example, through thebiosynthetic pathway.

5EM-Visualization at Ambient Temperatures

All EMs are rather sophisticated machinesbecause electrons can only be usefully

Fig. 13 High-pressure freezing and freeze-substitution. Shows freeze-substituted specimensfollowing high-pressure freezing. (a) Shows the nematode Diploscapter coronata having been vitrifiedwithin a capillary tube (the tube wall is indicated); the freeze-substitution was done using osmium inacetone. I-intestine; G, gonad (b) Shows a section through a high-pressure frozen andfreeze-substituted yeast cell (Saccharomyces cerevisiae). The specimen was embedded in LowicrylHM20 resin. Yeast cells are notoriously difficult to preserve by conventional fixation methods due totheir robust cell wall. This image shows remarkable preservation. The arrowheads indicate directcontinuities between the nuclear envelope and the endoplasmic reticulum. This ER, seen in negativecontrast, is continuous with a domain of ER that seems to be very closely attached to the plasmamembrane (arrowhead adjacent to the plasma membrane (PM)). The nuclear pores are also evidentin negative contrast (arrows). Mitochondria (M) and other organelles are also evident. Both imagescourtesy of Heinz Schwarz.

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(a)

FP

PM

200 nm

Fig. 14 Cryo- and plastic-section immunolabeling of African trypanosomes.These figures show the trypanosome Trypanasoma brucei. (a) and (b) showTokuyasu cryosections of this organism following labeling for the variablesurface antigen (VSG). The VSG is seen to be heavily concentrated at the PM,the flagellar pocket (FP, the flagellum is indicated by F), throughout the Golgicomplex (G), as well as in ER and endocytic organelles (not indicated). Thearrow in (a) shows a labeled endocytic vesicle in the process of budding fromthe flagellar pocket membrane. Bars–300 nm.

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Fig. 15 Cryo- and plastic-sectionimmunolabeling of Africantrypanosomes. These figures show thetrypanosome Trypanasoma brucei.(a) shows a trypanosome that was fixedin 0.05% glutaraldehyde/2%formaldehyde before being embedded inLowicryl HM 20 after the progressivelowering of temperature method. Thesections were labeled with anti-tubulinand donkey anti-mouse gold (12 nm).The array of microtubules beneath theplasma membrane and in the flagellumare strongly labeled (arrow). (b) Shows atrypanosome that was vitrified byhigh-pressure freezing followed byfreeze-substitution in 0.5% osmiumtetroxide, 0.5% gallic acid in acetoneand room-temperature embedding inEpon. The excellent preservation of themicrotubules is evident by the cleardelineation of the tubulin protofilamentsin cross-section. Micrographs courtesyof Christoph Gr

..unfelder, Peter Overath,

and Heinz Schwarz.

FP

N

200 nm

(a)

200 nm

(b)

transmitted in a rather high vacuum (bet-ter than 10−3 Pa ∼10−4/10−5 Torr). Thishigh vacuum in the main column alsoneeds to be maintained when a grid isintroduced. For this, the grid is first intro-duced into a low volume prechamber (air-lock) that is quickly pumped to vacuum.The grid can then be carefully insertedinto the center of the objective (electromag-netic) lens. At the top of the microscopecolumn is an electron source (tungstenfilament, lanthanum hexaboride crystal)or, the (most coherent and expensive)field-emission filament. The specimen is

observed at acceleration voltages rangingfrom 80 to 120 kV (standard) to 200 to400 kV (new ‘‘intermediate-voltage’’ mi-croscopes) or up to 1 million volts inthe high-voltage EM (of which only asmall number are available for biologi-cal research worldwide; the others havedisappeared due to lack of interest). The re-cent developments in EM tomography willlikely see a reversal of this trend, as meth-ods to investigate thicker sections becomemore useful (see below).

The emitted electrons are attractedtoward the anode leading to a coherent

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(a)

100 nm

100 nm

(b)

Fig. 16 Freeze-fracture immunolabeling. (a) Shows chick liver, tight junctionslabeled for the tight junction protein occludin while (b) indicates rat liver, gapjunctions double-labeled for connexin 32 (large gold particles), and connexin 26(small particle). The tissue slices were quick-frozen by contact with a copperblock cooled with liquid helium. The frozen samples were fractured in a BalzersBAF 400 T freeze-etch unit at −110 ◦C, replicated by deposition ofplatinum/carbon (Pt/C) followed by carbon. After thawing and washing withPBS, the pieces of Pt/C replica were transferred to 2.5% SDS containing-bufferfor 12 h at room temperature. Subsequently, the replicas were immunogoldlabeled, rinsed, fixed with glutaraldehyde, rinsed again, and picked up on grids.Micrographs courtesy of Kazushi Fujimoto.

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beam of electrons that, with the help of anelectromagnetic condenser lens, can thenbe converged onto the specimen. Electron-dense material (biological material heavymetal stains) scatter the electrons, whereasthe electron-lucent parts of the specimenallow the electrons to pass down thecolumn where, after magnification by theobjective and projector lenses, they forman image on a phosphorescent screen, filmor CCD camera. There are two differenttypes of interactions between electronsand biological material; elastically scatteredelectrons interact with the specimen witha minimum loss of energy and thereforethere is no change in the wavelength ofthe electrons. This type of scattering is dueto a simple deviation of the electrons inthe atoms in the specimen. In contrast,inelastically scattered electrons are thosethat experience energy loss and changeof wavelength during their interactionswith the electrons of the encounteredatoms. These interactions give rise tothe secondary events, such as heatingthe specimen, mass loss, contrast, X-rayemissions and cathodoluminescence.

When electrons interact with the phos-phorescent screen, the green light emittedby fluorescence provides a signal that thehuman eye sees and the brain needs to in-terpret. By removing the phosphorescentscreen transiently, the electrons can be di-rected toward photographic emulsion torecord the image on film. Cooled CCDcameras are now rapidly replacing filmbut they are very expensive and still do nothave the resolution available on film. It islikely to be only a matter of time beforeCCD cameras replace film completely.

The inelastically scattered electronspassing through the specimen can damagethe specimen, resulting, for example, inmass loss, even in plastic sections. However,in practice, beam damage is not a serious

problem for plastic or Tokuyasu cryosec-tions, at the usual level of resolutionneeded. For vitrified, native specimensthis is a very serious concern, as we willnow discuss.

6EM at Cold Temperatures

Imaging vitreous specimens, either bulksuspension, or hydrated cryosections, isa lot more difficult than imaging spec-imens at ambient temperatures. This isreally not something that can be taught ina manual. Moreover, one has to deal withquite delicate and expensive equipment.A competent cryo-EM specialist needs tounderstand a lot about the physics ofthe processes involved. Native biologicalsections are extremely sensitive to the elec-tron beam, and under the conditions usedfor ambient temperature specimens, theyvisibly boil away within seconds. This ne-cessitates low-dose imaging. In the standardapproach, the investigator can only sur-mise that suitable specimens are present ata site selected at low magnification, that is,immediately photographed at higher mag-nification using the minimum electrondose required for imaging. All focusingand image adjustments are therefore doneat sites away from the selected areas. Inrecent times, the technology available withmodern EMs has improved considerably,and is now much more user-friendly.

In most cases, the specimen is unstainedand to acquire sufficient contrast tosee the details is often a problem. Foroptimal images, one takes advantage of theincrease in phase-contrast that is providedby underfocusing.

Nevertheless, the fact remains that moreand more specialists are now routinelyrecording images of very high information

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content with this technology. There is nodoubt that at the top level the cryoimagingapproach provides the highest resolutionavailable in EM today (see Fig. 11). Insuitable specimens, by averaging datafrom large numbers of micrographs, itcan now approach (or reach) the atomiclevel of resolution (0.3–1 nm) when two-dimensional crystals are available. In manycases where a 1 to 3-nm resolution isobtained, the level of information oftensuffices to help one to fit higher resolutionX-ray crystallographic data of the samestructure into a comprehensive model ofthe structure. In this way, the details of the(high-resolution) X-ray structure are fittedonto its overall shape, which emerges fromthe (lower resolution) cryo-EM model.

It is important to realize that in order to‘‘solve’’ a structure to the desired level ofresolution, the availability of negatives oronline images is merely the beginning ofa long, complex, and often tortuous pro-cess. In contrast to ambient temperaturespecimens in which the images are mostly‘‘final,’’ in cryo-EM (as well as in somehigh-resolution negative stained specimenanalysis), there is an increasing number ofpossibilities to obtain much more relevantstructural information by use of compu-tational image processing algorithms. Thegoal of these procedures is to improvesignal-to-noise ratio by averaging a largenumber of structural units. The two mainkinds of approach to obtain this goal arethe following: (1) Electron crystallography(including helical reconstructions) of two-dimensional crystals and (2) Single particleanalysis, including icosahedral particles,where particles imaged at various orien-tations are collected, averaged and super-imposed into a three-dimensional model.

Examples of cryoimages obtained usingsections are shown in Fig. 12 and from

whole-mounts of particles in Fig. 11 and19(a) and of cells in Fig. 19(b).

6.1EM Tomography

In the past, if one wanted a three-dimensional view of a structure from thinsections it was necessary to use serialsections. The need to cut and maintainin perfect order up to a hundred or morethin sections is technically quite demand-ing and, until recently it was very difficultto subsequently align images from eachsection on top of each other in perfectregister. In the past few years, a revolu-tionary alternative approach has startedto have a major impact on EM at alllevels. From either a single particle ora relatively thick section, tomography al-lows one to obtain a three-dimensionalreconstruction of the structure of interest.In practice, a device for tilting the speci-men, the goniometer stage, allows the gridto be tilted relative to the beam (understandard conditions + and −∼70◦; newgoniometers are now reaching ±80◦). Im-ages of all the projections are collected,usually at intervals of 1 to 2◦. By ‘‘back-projecting’’ these images into a three-dimensional volume one can constructthe original image in three dimensionusing now standard computational algo-rithms. The reader is also referred to awhole volume of the Journal of StructuralBiology that was dedicated to the excit-ing developments in this field (Vol. 138,pp. 1–155, 2002).

There are a number of problems thatcan limit this approach. First, since thereare a large number of images, the effectsof beam damage have to be considered.In practice, this is not a serious problemfor the ‘‘ambient temperature specimens’’indicated above: one can take 130 to 140

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images, 1◦ apart. However, for vitrifiedparticles or sections, this problem is stilla limitation; in practice one is currentlyrestricted to, at most 100 images beforethe specimen is irreparably damaged.It should be emphasized that a greatdeal of effort has been made by afew specialists to make this technologyavailable, with important developments insoftware, automatic image collection andin specimen preparation.

The second problem is the ‘‘missingcone’’ or wedge due to the practicallimitation that one cannot tilt the grid±90◦ – there will always be a part ofthe information that is missed, resultingin loss of resolution. For quasi-regularparticles, one can overcome this problemby averaging a number of reconstructionsfrom different viewpoints. For imagingcomplex structures in sections of cells,it may be possible to combine tomogramsfrom sections that contain the specimen atdifferent orientations.

In addition to being used in high-resolution EM for reconstructing singleparticles such as ribosomes this approach,in combination with high-pressure freez-ing and freeze-substitution, is now beingapplied to analyze the three-dimensionalorganization of different structures, suchas whole yeast cells, the Golgi complex,mitochondria (see Fig. 18). Most of thesestudies have used relatively thick (−200−300 nm) plastic sections. It should benoted that for the difficult question of de-ciding whether or not fine tubules connectcertain structures (notably the Golgi com-plex), this approach can still leave openconsiderable room for subjectivity. In suchcases, the use of heavy metal stains (e.g.the cytochemical reaction product of HRP,or other enzyme reaction products) to fillthe lumen of tubules (e.g. by expressing

HRP-containing constructs in cells) cangreatly aid the analysis.

The ability to visualize whole cells directlyby a transmission cryo-EM approach canbe considered the ultimate goal in electronmicroscopy. It is now fascinating toconsider that, prior to the developmentof robust methods for preparing thin resinsections of cells, the famous trio of Porter,Claude, and Fullam had made initialattempts in 1945 to visualize cells grownon EM-grids (after osmium tetroxide vaporor formaldehyde primary fixation). Despitethe harshness of the preparation (bytoday’s standards), what was observedwas striking. In the thinner, peripheralregions of the fibroblast, whose outlinesare clearly delineated, one can clearly seemitochondria, filaments and so on (•see Q14

Fig. 17).In an important development, the

Baumeister group have succeeded inbringing together a whole battery of techni-cal developments to enable the experimentto be redone with state of cryo-EM tomog-raphy. In this publication, they focusedon the amoeba, Dictyostelium, that in ac-companying videos could be seen to moverapidly across the grid just before vitri-fication. Although one has to accept the(still significant) limitation that only cellprojections thinner than ∼400 to 500 nmcould be clearly analyzed, the details shownare amazing, at a resolution of ∼5 nm. Itmust be realized that this is the first timeit has been possible to look directly in-side a relatively unperturbed cell at thislevel of resolution. An example is given inFig. 19(b).

With more developments, and morewidespread use of this approach, (likelyaided by higher accelerating voltages andenergy filtering) thicker regions of thecell will become more accessible foranalysis. The use of hydrated cryosections

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N

1 µm

Fig. 17 One of the first micrographs ofcells. Shows a famous micrograph takenfrom the paper by Porter, Claude, andFullam from the Journal of ExperimentalMedicine (1945). This micrograph isone of the first ever taken by electronmicroscopy. The cells were grown on anEM grid, fixed in osmium tetroxide,air-dried, and visualized as a wholemount. Micro spikes (arrow) at theleading edge are evident, as is thenucleus and the worm-shapedmitochondria. A fine reticulum beneaththe mitochondria is likely to be theendoplasmic reticulum. This image hasalready been shown in a wonderful bookby Christian de Duve in 1984.

can be expected to complement theseefforts. The ability to reconstruct detailedstructures (e.g. within the nucleus) willgreatly increase the level of informationone can obtain from these preparations.Tomography can also greatly simplify theinterpretation of the three-dimensionalstructure from hydrated cryosections.

6.2Low- and High-resolution SEM

The ability to visualize surface structuresby scanning EM is well known, even tomany nonscientists, a result of spectacularimages of animals, plants, and microor-ganisms obtained by this approach. Manyspecimens, such as insects have robust

exoskeletons that can withstand beamdamage and only a thin layer of metalneeds to be evaporated before visualiza-tion; for a beautiful example see Fig. 20.This low-resolution SEM approach methodis widely used. At a higher level of resolu-tion, parts of the organ of Corti of the earare seen in Fig. 21.

Over the past 20 years, the use of higherresolution SEM in molecular cell biol-ogy has dwindled – only a handful oflaboratories currently use this approach.This is indeed surprising, and very disap-pointing given the proven ability of thisapproach to see high-resolution details ofstructures such as nuclear pores, bacterio-phages (Fig. 22(a, b)) and actin filaments(Fig. 22c).

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−60 0 +60

(a)

(b) (c)

(d) (e)

Fig. 18 EM Tomography from plastic thick sections. (a) To obtain a three-dimensionalreconstruction of an organelle, a tilted-series of images is recorded. A 250-nm section ofmultilamellar lysosomes in a mouse dendritic cell is shown after high-pressure freezing,freeze-substitution, and resin embedding. This, relatively thick section was tilted from −60◦to 60◦ along two perpendicular axes to produce a tilt-series of 242 images. This panel showsthree of these images, collected at −60, 0, and 60◦s tilting. The small black dots in theimages are 10-nm gold beads on top of the section, which are used as fiducial markers(reference structures) for the three-dimensional reconstruction. (b) After calculating thethree-dimensional reconstruction from the tilt-series, the three-dimensional volume can beanalyzed by displaying it as thin, so-called tomographic slices. The slices can be made alongevery possible axis. Three slices through the tomographic volume are shown: top: X-Z,middle: X-Y and right: Y-Z. (c) By manually tracing the membranes of the multilamellarlysosomes in the tomographic volume a three-dimensional model is created. (d) Shows athree-dimensional model top view of one of the tomographic slices, while (e) reveals thethree-dimensional model view of the endocytic vesicle. The figure was provided by Jean-LucMurk and Bruno Humbel. From a study by Murk et al., J. Microsc. in press.

In conjunction with immunogold la-beling prior to vitrification, the use ofcryo-SEM, especially the remarkable newgeneration of field-emission (FE) scanningelectron microscopes offers the molecular

cell biologist a complementary approach totomography. SEM visualizes the surface ofthe structure directly in three dimension.In conjunction with the pioneering effortsof a few specialist groups (especially those

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of Martin Muller, Paul Walther, Hans Risand Terry Allen) in developing new ap-proaches for specimen preparation, thismethod is now capable of extremely high-resolution (1–3 nm) (Fig. 22). A numberof publications have shown the greatpotential of this approach, also for im-munogold labeling, especially when goldparticles as small as 1 nm are used, thatcan be visualized in the backscatter mode.

In some studies, Fab domains have beenused instead of whole IgG molecules toincrease the precision of the labeling.

6.3Critical Point Drying

The most common method to prepareliving specimens for SEM has beenchemical fixation followed by critical point

(a)

100 nm

100 nm

VV-Int

Core

ER

Actin

Actin PM

R

MT

(b)

Fig. 19

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Fig. 20 Classical SEM. Shows abeautiful image of Drosophilamelanogaster visualized by conventionalSEM. The insect was fixed withglutaraldehyde, rinsed,critically – point-dried and sputtercoated using gold-palladium. Finedetails of the surface of the insect canbe seen in artistic detail. Micrographcourtesy of J

..urgen Berger (Max Planck

Institute for Developmental Biology,Tuebingen, Germany).

drying (CPD). The basic idea of this ap-proach is to prevent drying artifacts, whichoccurs when the sample is exposed tothe surface tension of the liquid–gaseousphase border. This method has also beenextensively used for preparing material forTEM replicas and for epoxy resin embed-ding. For this, the specimen is chemicallyfixed and dehydrated in solvents such asethanol (essentially as for epoxy resin em-bedding). Later the solvent is replacedby liquid carbon dioxide in the pressurechamber of the critical point drying device.Then, the carbon dioxide is slowly warmedup. This causes the pressure to rise in the

closed system. When the temperature andpressure pass above the so-called ‘‘criticalpoint’’ of carbon dioxide (31 ◦C and 74 bar),the pressure is carefully reduced and thesample removed once room pressure isreached. The specimen is thus dried with-out ever having seen the liquid–gas phaseborder, which can cause major distortionsto a sensitive specimen. Recent applica-tions of the CPD method in cell biologyare the preparation of cytoskeleton ele-ments after detergent treatment and TEMreplica. As pointed out by Ris, it is very im-portant to remove every trace of water fromthe specimen before the next stage, which

Fig. 19 Cryo-EM and tomography. (a) Shows cryo-EM of vitrified vaccinia virus using the bare-gridmethod. This virus is enormously complex as was already evident in Fig 4. Although a tremendousamount of information is present in these particles, at present the detailed three–dimensionalorganization of the virus remains to be deciphered and the tomographic approach is now in progress.(b) Shows the 2002 equivalent of the experiment already carried out. Hela cells were grown onEM-grids and vaccinia virus was added. The goal of this experiment was to visualize intermediates inthe entry of the virus into the cell, a highly complex and not understood process. The grid was vitrifiedand examined by cryo-EM. This image is a section through a tomogram (not a physical section)showing details that are not approachable by single projections. The upper particle is, we believe, anintermediate (from VV-Int) in the entry of the viral core and is likely to be still outside the cell. Incontrast, the viral• core (c) indicated, which is transcriptionally competent, having lost its outerQ8membranes is definitely within the cytoplasm. Actin filaments are seen in the lower part of themicrograph, whereas the filaments above the core are microtubules. Ribosomes (R) are also evident,as is the ER. This method is the first approach available for looking inside physically and chemicallyunperturbed cells by EM at this level of resolution. Bars = 100 nm. Both micrographs courtesy ofMarek Cyrklaff; specimen provided by Jacomine Krijnse Locker.

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1

2

3

10 µm

Fig. 21 SEM of the organ of Corti of themouse ear. These SEM images show aguinea pig cochlea dissected to removeHensen cells (sensory cells of the earand the supporting cells). Hensen cellswere dissected to give a clear view of theouter hair cells, the sensory cells of theear (2) and supporting cells. The tops ofthe outer hair cells have stereociliaattached ((3) the chevron-shapedprojections) and the main body of theouter hair cell can be seen below. (1)indicates the outer phalangeal cells ofDeiter. The specimen was fixed inglutaraldehyde before dissection andthen infiltrated with tannic acid andosmium tetroxide. This facilitatedexamination without metal coating in afield-emission SEM (a Philips XL-30FESEM operating at 5 kV). Thisspecimen preparation method was amodification of the method ofJongebloed et al. (1999). Micrographcourtesy of Paul Webster.

for SEM involves coating the specimenwith a thin layer of metal (•see Fig. 23).Q15

6.4Freeze-drying and Cryo-SEM

The SEM is able to image the physicalsurface only. In most biological samples,however, the structures of interest are in-side cells and tissue. To get access tothe structures of interest, the specimenhas to be opened by cracking or by sec-tioning, or the overlaying water must beremoved. This is only possible with a solid-state sample. The most straightforwardway to solidify a biological sample is byvitrification. In the cryo-SEM, bulk sam-ples can be investigated over a very largerange of magnification, from the resolu-tion level of a dissection microscope upto macromolecular resolution. After beingimmobilized, by cryofixation the samplescan be investigated in the cryo-SEM in

the fully frozen-hydrated or in the partiallyfreeze-dried (‘‘deep-etched’’) state. Manyartifacts of conventional chemical fixationand dehydration techniques can, thereby,be prevented.

Removal of the vitrified water is pos-sible by sublimation in the vacuum inthe microscope or in a preparation cham-ber. This preparation step is also called‘‘freeze-etching’’ with a partial sublimationof the water, or freeze-drying in the cryo-EM literature. When the water is partiallyremoved, the three-dimensional arrange-ment of the structures in the cytosol canbe investigated. Current methodologicalresearch focuses on a better control of theice-sublimation process.

This method also has a high, and stillnot fully used potential for the analysis ofin vitro systems in cell biology. Togetherwith Paul Walther, our group is currentlyinvestigating actin-membrane interactionsusing these cryo-SEM approaches.

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(a)

(b)

100 nm

100 nm

(c)

10 nm

Fig. 22 High-resolution cryoscanning EM. (a) and (b) show theT-even bacteriophage Tull ∗ −46 after freeze-drying, metal coatingand visualization by cryofield emission SEM. (a) shows an intactparticle, whereas (b) is a particle that became disrupted byspesimen preparation. (a) is additionally labelled with an Fabdirected towards the tail protein Y of the phage. Individual Fabmolecules can be seen decorating pieces of the isolated tail(small arrows) as well as the helical ribbon of the tail protein. In(b) following disruption, the DNA of the phage is clearly evident(large arrow), as are many other structural details of the innerparts of the virus. (c) shows high-resolution image of an actinfilament that had been freeze-dried and coated withtantalum-tungsten. ln(a) and (b) by courtesy of Rene Herman,Heinz Schwarz, and Martin Mueller. (c) Courtesy of Paul Walther.

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(a)

100 nm

100 nm

(b)

100 nm

(c)

Fig. 23

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6.5Coating Techniques for Cryo-SEM

Samples for high-resolution SEM areusually coated with a thin metal layer inorder to enhance electrical conductivityand to localize the signal on the surfaceof the sample. Charging of the samplecan also be reduced by working at lowaccelerating voltage of the primary beam(e.g. 2 kV). It was, however, pointedout, that the excitation volume of thesecondary electrons is increased whenthey interact with an insulator. Therefore,it is advantageous to work with coated(shadowed), conductive samples, evenwhen working at low voltages. Relativelythin samples can ideally be supportedby a uniform layer of an electricallyconductive material, for example, by 1 nmof tungsten. Bulk cryofractured samples,however, are insulators and very beam-sensitive. For these samples, double layercoating has been very useful. The sample isfirst coated with a contrast-forming layerof heavy metal (platinum or tungsten),both to enhance electrical conductivityand for mechanical stabilization. A carbonlayer with a thickness of 5 to 10 nmis then applied on top of the platinum

layer. As shown in different studies, thislayer drastically reduces the effects ofbeam damage. For the analysis of thesesamples, the backscattered electron signalhas to be used, which is mainly producedby the metal coat that stays in directcontact with the biological structure ofinterest.

6.6Scanning Transmission EM

A specialized variation of transmissionEM is scanning transmission electronmicroscopy (STEM, done mostly in thedark-field mode) in which the specimenon the grid is scanned in a raster patternwith a finely focused electron beam.This offers precise determination of theabsolute mass of the specimen irrespectiveof shape.

6.7X ray Elemental Microanalysis by EM

X-ray microanalysis of biological speci-mens can be conducted at two differentlevels of resolution. Low-resolution mi-croanalysis is typically performed usinga scanning electron microscope equipped

Fig. 23 Metal shadowing for EM. (a) Shows the identical TMV particles that were seen by negativestaining in Fig. 3. This unpublished image, over 30 years old, was unidirectionally shadowedwith platinum. (b) Unidirectional surface shadowing (tantalum/tungsten, elevation angle −45◦) of amicrotubule stabilized with small amounts of tau-protein. This protein is essentially invisible due toits highly filamentous structure. This leaves a surface pattern typical for microtubules, exhibiting theouter protofilament surface as a continuous axially oriented rim. The arrow shows a protofilamentsplayed out at the end of the microtubule. (c) Unidirectional surface shadowing of tubulin thatcoassembled with walls• monomeric kinesin molecules in vitro. The presence of these moleculesQ9renders the outer surface in a completely different way relative to the undecorated microtubules in(b). Instead of an axial striation, one now observes a perpendicular striation with an axial 8-nm repeat(arrows), which corresponds to one motor domain bound to each α, β-tubulin dimer. This methodallows one to clearly distinguish between inner and outer microtubule surface. The inner surfaceexhibits a 4-nm repeating pattern according to each tubulin monomer. (a) Courtesy of Herman Frankand Heinz Schwarz; (b) and (c) courtesy of Andy Hoenger.

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with an X-ray detector. This applicationis typically used to analyze small particu-late inclusions in cells and tissues. Theanalysis of environmental heavy metalcontamination, foreign body ingestion,and occupational exposure are the mosttypical applications. Examination of cellsand tissues in the scanning electron mi-croscope using the backscatter electronimaging mode can also be used to providelow-resolution information, for example,inclusions and particles containing highatomic number atoms can be identified inassociation with particular cell types.

X-ray microanalysis of biological spec-imens can also utilize a transmissionelectron microscope coupled with an X-ray detector, but the demands of speci-men preparation and specimen handlingare much more challenging. Typically,this high-resolution technique requiresthe rapid freezing of small, undamaged

biological specimens. The biological speci-men requirement involves selecting small(less than 1 mm) specimens, which mustbe solidified using ultra rapid freezingtechniques leading to vitrification. Subse-quently, the small specimens are mountedin a cryoultramicrotome using either me-chanical clamping or a low temperaturecryoglue. The ultrathin sections with thick-nesses of about 100 to 120 nm are pre-pared and transferred to formvar-coateddouble-folding (sandwich) grids in the cry-ochamber of the cryoultramicrotome. Thegrid is next transferred to a cryotransferstage at −170 ◦C. The cryotransfer stageis inserted into the transmission electronmicroscope. The transmission electron mi-croscope must be equipped with a liquidnitrogen cooled anticontamination deviceto prevent the deposition of water vaporonto the specimen from the column of theelectron microscope.

a1 a2 a3 a4

Position of the first plane uniform randomin [0,k], estimate of volume = k(a1 + a2 + a3 + a4)

k

Point counting

d

N

(a) Intercept counting

d

(b)

(c) Fig. 24

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For X-ray microanalysis, the specimenmust be warmed to about −100 ◦C, whichallows the water in the specimen to sub-lime onto the anti contaminator. Thisfreeze-drying step generally requires 45to 60 min to achieve a specimen thatwill withstand the high-beam currentsnecessary for high-resolution X-ray mi-croanalysis. Using this technique allowsthe analysis of subcellular regions inindividual cells such as mitochondria, lyso-somes, cytoplasm regions, and nuclearregions.

The critical assumptions in this tech-nique are (1) the freezing process doesnot translocate diffusible elements fromtheir native location; (2) that the cryosec-tioning process and freeze-drying of theultrathin section in the electron micro-scope also do not translocate diffusibleelements from their native sites, and(3) that during analysis under the high

electron beam current the diffusible el-ements also remained in their nativelocation. In practice, these criteria arerarely fulfilled for small, mobile ionsof cell biological interest, such as cal-cium or magnesium. In general, thesetechnical difficulties have limited theapplications of this technique in cellbiology.

An additional limitation is the lack ofsensitivity to small changes in concen-trations of sodium, chlorine, potassium,and calcium in the small subregions ofcells. Under optimal conditions usingfreeze-dried thin sections, the limit of de-tection for X-ray analysis in general isabout 100 parts per million. It is verydifficult to estimate the water content ofthe subcellular regions so that the dryweight concentration measurement canbe converted to a wet weight concen-tration that is meaningful to biologists.

Fig. 24 Principles of Stereology. (a) Shows the principle of estimating area using point counts. Atransparent lattice grid is randomly positioned over the images of a structure, in this case, ahypothetical profile through a cell, the nucleus N is shown. The point where the edges of the two linesof the lattice meet each other is considered to be a unique point (arrows). One simply counts thenumber of points over the structure of interest. In this particular example, there are 13 points in totalover the cell, four of which follow the nucleus. If this result were seen after averaging over a numberof micrographs, this means that the nucleus represents 4/13 = 30.7% of the cell volume. Thenumber of points P × d2 provides an estimate of the area of each structure on the micrographs. (b)Shows the principle of intercept counting to estimate the boundary length of surface. In this example,a square lattice grid with random orientation and position is placed over a piece of string. The arrowsindicate the positions at which the lines of the string transect the lines of the grid: These positions(formally defined by the intersection of the edges of the line and one edge of the string) are referredto as intercepts. The length of the line is then given by π/4 × I × d where I is the number ofintercepts and d is the distance between the lines of the lattice grid. This principle, first introduced byBuffon, is an unbiased estimator of the length of the profile, and can be converted to surface area inthree dimensions. In this example, if d = 1 cm and there are 21 intercepts, then the length of thestring is π(3.142)/4 × 21(Intercepts) × 1 cm = 16.495 cm. (c) Shows the principle of the Cavalierimethod. The principle here is to section a three-dimensional object with a series of equally spacedsections such that the position of the first section is random within the interval between the sections.In this example, four sections are made, a1–a4, and the volume is then given by the sum of the areasof the object displayed on the sections multiplied by the distance (k) between the sections (sectionthickness). This method is justifiably considered to be a powerful method for stereology, and hasbeen widely used for many different organs, tissues, cells, and organelles. Diagrams courtesy ofJohn Lucocq.

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Theoretically, the water content could beestimated by making measurements be-fore and after freeze-drying; however, fullyhydrated specimens in the electron mi-croscope are intolerant of the high-beamcurrents that are necessary for X-ray mi-croanalysis.

In summary, the most useful methodsinvolving X-ray microanalysis at presentare performed in the scanning electronmicroscope at low resolution for studyingoccupational exposure, mineralized tissue,and calcification associated with diseaseprocesses.

6.8Energy-filtering Transmission ElectronMicroscopy (EFTEM)

This approach allows improved imagingof thick sections and frozen-hydratedspecimens by removing inelasticallyscattered electrons. The amount ofinelastic scattering increases withspecimen thickness (in general quiterapidly) and is dependent on theincident electron energy and on thematerial the beam is interacting with(in general, the heavier the material,the stronger the interaction). In EFTEM,the electrons are separated according totheir energies, making it possible tofilter out the blurring effects seen withthick sections, as well as retrieve selectedchemical or physical information fromthe specimen. This additional electronselection results in contrast enhancementfor all imaging modes and also providesthe possibility of selecting electrons withspecific scattering effects for imaging,thereby generating object- or element-specific contrast. For more details asprovided by one of the manufacturersof this technology, see (www.leo-em.co.uk/temproducts/principle.htm).

7Stereology

Although many cell biologists have yet torealize it, a remarkable set of methods hasbeen developed over the past 30 years forquantifying the sizes, contents, and spa-tial relationships of biological structures.These methods are known as Stereology,a rigorous, probability-based disciplinefor estimating three-dimensional struc-tural quantities (including volume, surfacearea, length, and number) and spatial re-lationships from sectional or slice images.The methods are design-based, meaningthat they rely on the principle of randomsampling (especially systematic randomsampling) and offer the twin benefits ofno (or minimal) bias combined with highprecision. The work needed to implementthem is, surprisingly, less than generallyexpected, so these methods tend also to behighly efficient. The use of stereologicalmethods will be outlined, as will recentlydeveloped approaches for quantifying im-munogold particles that are used to detectspecific antigens.

The sine qua non of stereology is theapplication of geometrical probes forestimating two-dimensional and three-dimensional quantities. Crucial for thisgoal is unbiased sampling at all levelsof specimen preparation and imaging:each specimen (e.g. an organ), and eachpart of that specimen (e.g. its differenttissue compartments), must have an equalchance of being sampled. For somequantities, it is necessary to randomizethe orientation of sectioning as well assection position. A number of books andchapters cover these important aspects ingreat detail. In order to give the flavor ofhow stereology works, we shall considerone method available for determining theabsolute volume of any structure and

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two standard methods for determiningrelative surface area (or length of sectionalmembrane image) and relative volume (orprofile area of sectioned structures). It willbe seen that, once the absolute volumeof a reference structure (e.g. the nucleusor the cell) is known, it is possible tocalculate the absolute total surface andvolume of any compartment in the cellusing their estimated relative surfaces andvolumes. It is equally easy to obtain higher-level quantitative information about cellsin tissues, organs, or whole organisms.

7.1Relative Area and Volume

In order to estimate the areas of the sec-tional images (profiles) of different struc-tures on micrographs, transparent plasticsheets having a uniform array of ‘‘testpoints’’ are randomly superimposed overthe image (see Fig. 24a). The positioningof the overlay with respect to the spec-imen must be randomized. These days,this can also be conveniently done by com-puter. Since each point corresponds to aprecise area, the total sectional area ofany structure is then directly proportionalto the average number of points fallingover it. For a set of micrographs, the testpoints are summed and averaged; relativeareas can be equated with relative volumes.The relative numbers of test points fallingon different structures within the speci-men then provide unbiased estimates ofthe relative profile areas of these struc-tures.

The relative point totals (and relativeareas) may be converted into relativevolumes (volume densities, volume frac-tions, or volume proportions) using theprinciple first described by the geologist,Delesse, in 1848. If the micrographic im-ages themselves represent a set obtained

from randomly positioned sites through-out the specimen, the relative numberof test points on each structure is notonly an estimator of their relative pro-file areas but also an estimator of theirrelative volume in the specimen (in fact,the points then sample three-dimensionalspace). This simple and efficient point-counting approach therefore allows thepossibility to characterize a set of cells interms of their relative contents of nucleus,cytosol, mitochondria, Golgi complex, andany other compartments of interest.

7.2Relative Profile Length and Surface Area

There is a simple but precise method forestimating the length of a linear trace(line on a sectional image), irrespectiveof its shape or orientation. On the basisof the principle first observed by Buffonin the nineteenth century, a system ofparallel test lines is randomly laid overthe profiles of interest. The lines must berandomized in terms of both their positionand orientation. This principle can besimply illustrated with a piece of string,which is crumpled (so as to randomizeits orientation) and then laid flat ona system of test lines (Fig. 24(b)). Thenumber of times the string is intersectedby the test lines (separated by distance,d) is counted and provides a simple andunbiased estimation of the length of string.If the lines on the overlay run in both‘‘horizontal’’ and ‘‘vertical’’ directions, thelength of the string is estimated bythe formula π/4 × I × d, where I is thenumber of intersections and d is thedistance between the lines of the grid(Fig. 24b). It is easy to visualize the lineartraces of membranes on TEM thin sectionsas the equivalents of pieces of string.

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By an analogous three-dimensional ap-proach based on randomly oriented en-counters between section planes and cellor organelle membranes, the relative num-ber of intersections between test lines anddifferent membrane compartments allowsone to estimate their relative surface areas,that is, surface of membrane per volume ofreference space. This simple and efficientintersection counting approach thereforeallows the possibility to characterize cellsin terms of the surface areas of their dif-ferent membranes.

7.3Absolute Volume

Sometimes it is not possible to estimatevolume by, say, Archimedes principle ofliquid displacement or by weighing, andit is necessary to resort to sectioning. Anelegant method for estimating the volumeof any structure by sectioning it wasinvented by Cavalieri in the seventeenthcentury. It was somehow forgotten untilit was rediscovered and was given moregeneral applicability as a stereologicalprinciple. The essence of the method issimple. First, the structure is sectionedcompletely into a uniform random setof slices (Fig. 24c). One can analyze eachsection but, as already mentioned, a greatstrength of stereology is the concept ofsampling. One systematically selects asubset of (e.g. 1 in every 5) sections.On each sampled section, the area isestimated by, say, the test point–countingprocedure. The combined area of all thesampled sections is then multiplied by themean distance between selected sectionsto obtain the Cavalieri estimate of volume.When proper sampling procedures arefollowed, this is a remarkably efficientand precise estimator of volume, atscales ranging from a lysosome to a

human brain. It can produce volumeestimates with a precision of 5%, or less,from just 5 to 6 sections chosen in asystematicrandom fashion.

7.4Surfaces and Volumes in Practice

Using the above principles for estimatingrelative surfaces and volumes, it is easyto appreciate how one could estimate theabsolute surface or volume of any cellstructure that is larger than the averagesection thickness (for technical reasonssmaller structures are problematical bythese methods). For this, one needs toknow one quantity in absolute terms. Formost• cells, biological questions that refer Q16

to quantity would pertain to the averagevolume of the cell (or its nucleus). TheCavalieri principle can be used to estimatecell or nuclear volume by either LM orEM. A rather simple method is to stain cellnuclei with a DNA dye, such as Hoechstdye or Dapi. By confocal microscopy, onecan optimally section through a set ofnuclei and, knowing the distance betweensections (given by differences in thefocusing plane) and the areas of sections(as above), one can estimate the averagenuclear volume by the Cavalieri principle.It is also possible to use 0.5 to 1 µm(semi-thin) section by EM (most nucleiin cells are 10 µm or less in diameterso a relatively small number of sectionssuffice). At 0.3 µm (or less), the procedurecan be done by EM.

Knowing the absolute volume of thenucleus, one would next take randomsections at the EM level. For volumeestimation, only the position of thesections relative to the specimen needs tobe randomized but, for surface estimation,both the position and orientation of thesections must be randomized. At a fairly

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low magnification, the overlay of testpoints can be used to tell us the relativevolume of the nucleus to the whole cell.For example, if on average 40 pointshit the nuclear profiles and 160 pointshit the cytoplasm, the nucleus occupies40/200 or 20% of the cell volume. If thenuclear volume is known (from above), theabsolute volume of the cell is five times thatof the nucleus.

The volume of any subcellular compart-ment of interest can now be estimated bymultiplying its volume ratio in the cell (VV )by total cell volume. Similarly, any surfaceof interest can be estimated by relatingthe number of intersections hitting the(membrane) profile of the compartment tothe number of points that fall on the en-tire cell. This gives us the ratio of surfaceof that membrane to the volume of thecell (SV ). Since S = SV × cell volume, thisgives an indirect but precise estimate ofthe absolute surface of the compartment.

There are also ingenious methods (againfrom Gundersen) for estimating particlenumerical density (number in a referencevolume) or total particle number. Numer-ical density is estimated by the dissectormethod, which relies on analysis usingtwo consecutive sections and an unbi-ased counting frame. If the referencevolume is known, an indirect estimate ofnumber is obtained by multiplying thisreference volume by particle numericaldensity. However, number can sometimesbe estimated directly using the fractionatormethod. These two approaches have rev-olutionized the ability of investigators toprovide unbiased estimates of the num-bers of cells in different tissues, mostprominently in various parts of the brain.

Using the test point and intersectioncounting principles described above, sim-ple methods are available for quantifyingimmunogold labeling. A major advantage

of the most recent developments is theability to compare observed distributionsof gold particles with predicted distribu-tions obtained by counting test points(organelle compartments) or test-line in-tersections (membrane compartments).The idea here is to use the uniform dis-tribution of the points and intercepts asindicators of randomness. Their predicteddistributions correspond to the patternsexpected for random labeling. Comparingobserved and predicted distributions al-lows one to identify compartments thatare preferentially labeled and to obtainestimates of their labeling densities andrelative labeling indices.

A number of stereology courses are of-fered regularly in Europe and in the UnitedStates and the interested reader is well ad-vised to attend such a course. Stereologyis not only a set of methods; it offers acompletely rigorous way of thinking aboutgood study design in cell and tissue biol-ogy. For quantifying structural parametersor for immunolabeling, the use of stere-ology should be standard. What is mostsurprising (given the relative lack of itsapplication for cell biology) is that thestereological approach can be so rapidlyand efficiently applied.

7.5Correlative Light and Electron Microscopy

It is not widely appreciated that sectionscut for immunolabeling at the EM levelcan also be very useful tools for label-ing at the LM level. This is true forplastic sections such as Lowicryl and forTokuyasu-thawed cryosections. Since thelabeling is restricted to one thin layeron the section surface, this approach canprovide better resolution than confocalmicroscopy. Moreover, multiple fluores-cence labeling with different antibodies on

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different successive serial sections is pos-sible; since each section (or small numberof consecutive sections) is labeled inde-pendently there is no restriction on thecombinations of antibodies used, for exam-ple, three different mouse antibodies canbe used, each identified using a different,distinguishable fluorochrome. DNA stainssuch as Dapi are also very useful sincethey can be used to find the thin sectionsby light microscopy. Since the sections arethin, a series of sections covers the samestructures and the images from the differ-ent antibodies can usually be combined togive pseudo-multiple labeling.

Recently, an elegant method has beendeveloped that allows visualization of GFP-labeled structures in a living cell and thenallows to identify the same cell (using anHRP-based approach) at the EM level.

7.6Interpretation in EM

Seeing and understanding somethingin three dimension is infinitely easierthan trying to interpret two-dimensional(section) images. Thin sections of cellsare highly deceptive because they mayseduce the investigator into thinkingthat the inside of the cell is opento a three-dimensional interpretation. Inprinciple, it is, but the fact remains thatit takes many years of looking down themicroscope before one can be considereda professional. The loss of the thirddimension is a more serious problem thanis often imagined.

An example was provided of a wellknown and highly accomplished electronmicroscopist, who misinterpreted the earlythin section images of the ER-cisternae asa system of parallel fibers. This exampleis useful because a large number of (evenexperienced) cell biologists automatically

think they see a thin ‘‘tube’’ (in 3D)when they visualize profiles of cisternae(especially when the profiles are rela-tively small).

Another significant challenge facing thebeginner in interpreting thin sections ishow to average information over many cellprofiles, no two of which can be consideredreally identical! Biases are rampant in thefield as one or a few ‘‘representative’’micrographs are selected for publication.This is especially a problem in presentingimmunolabeling images in the absence ofquantitation.

7.7Which Technique for Which Question?

Faced with all of these different possi-bilities for preparing specimens for EM,the beginner may have difficulty decidingwhere to start. The first advice is to con-tact a specialist laboratory. Much will thendepend on the scale, whether the startingspecimen is isolated vesicles, cells, tissues,or an organism, whose size can scale froma virus or bacterium to a whole organ-ism, such as an insect. Starting at thehighest scale, the surface of an organismless than about 1 mm can be visualizeddirectly by SEM. Above this size, the spec-imen must be physically cut at the firstpreparation step. An organism as large as1 mm can be seen at low resolution byconventional ambient temperature SEMs.For higher resolution, the specimen mustbe available as sets of ‘‘particles,’’ wholecells, or sections of cells or tissues. In thiscase, a cryobased specimen preparation ismostly obligatory.

For TEM, particles smaller than 0.3 to0.5 µm in height can be directly viewed bycryo-EM or by on-grid negative (positive)staining. The latter should always be thefirst approach for any such specimens

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since it can be done in 5 min with simpletechnology. Freeze-fracture methods canalso be applied for isolated particles,especially to see the inner details ofmembranes in a clearer fashion ratherthan in the much more limited cross-section profile evident in thin sections.However, the majority of EM studiesinvolve the use of such sections in order tosee the detailed ultrastructure within cells.The key question is which approach doI use for (1) structural questions, and (2)immunolabeling questions in the contextof ultrastructure.

For purely structural questions for bestresults, the combination of high-pressurefreezing and freeze-substitution is recom-mended. The hydrated cryosection methodmay be preferred for some questions whenone has access to this technology. If one ac-cepts a potentially lower level of specimenquality one can select one of the two sec-tioning approaches, plastic embedding orTokuyasu cryosectioning. Which approachis selected is largely a question of taste; atthe top level, the results are often very simi-lar, although the Tokuyasu method has theadvantage over conventional embedding inthat it avoids solvent dehydration.

For immunolabeling the first questionis preembedding versus on-section label-ing. Preembedding using immunogoldis highly empirical and time-consuming,compared to section labeling. However,when it works, one has the advantage thatone has the labeled specimen already em-bedded in (usually) epoxy resin, which canbe cut with standard ultramicrotomes thatare widely available. When the labelingis not successful, there is a significantlylarger number of variables to adjust thanwith on-section labeling.

If the antigen is within the lumenof a membrane compartment, such asthe ER, it makes little sense to try

conventional immunogold preembeddinglabeling because of the difficulties of theantibodies obtaining access to these sites,unless one decides to try to completelyopen these structures by some means.HRP-based methods can be used better,but one has to accept the fact that thisapproach is not quantitative and can evenbe quantitatively misleading.

For the majority of localization ques-tions, on-section labeling is the methodof choice. In this approach, each part ofevery structure has, in theory, an equalchoice of being expressed to the antibodyon the section surface. In practice, how-ever, the situation is more complex. Animportant question is whether one shoulduse a plastic-section approach (Lowicryl,LR White etc.) or the Tokuyasu cryosec-tion method. The latter method is fasterand recommended for antigens that areless likely to be extracted or artificially mis-localized during the section pickup stage;such artifacts are especially a danger withsmall, soluble molecules and lipids. It isgenerally an excellent method for mem-brane proteins and cytoskeletal proteins;since there is no embedding medium perse there is generally more access of anti-bodies to antigens in the section than isthe case for plastic sections, where accessis strictly surface-limited. While plasticsections tend to label less than cryosec-tions, they have the advantage that thepolymerized resin better ‘‘fixes’’ the anti-gen against redistribution.

In conjunction with freeze-substitution,the low temperature Lowicryl resins offerthe ultimate level of specimen preparationthat is compatible with immunolabeling.For small diffusible molecules and lipids,this is absolutely the method of choice.When all methods fail, even with anti-bodies that give a good signal by lightmicroscopy, the two most likely reasons

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are, first, lack of accessibility of the anti-body to the antigen, even on the surfaceof the section, and second, the concen-tration of available antigen on the sectionsurface is simply below detection. A sen-sible approach is to use similar sectionsfor immunofluorescence analysis that areused for EM; this approach is likely to bemore sensitive.

7.8Final Comment

Given the breadth of techniques avail-able, it is somewhat surprising that theuse of more of these EM methods isnot sufficiently widespread. Rather thanbeing standard methods in every cell bi-ology institute worldwide, the number ofspecialist groups has been declining (ex-cept for the bare-grid cryo-EM method,which is increasing in use). This prob-lem, and the politics and sociology behindit have recently been discussed in detail.The best possibility to learn these tech-niques is to visit specialist groups. Inaddition, practical courses are given inmany countries, often sponsored by na-tional EM societies. We teach an annualEMBO-sponsored course in Europe everyyear. For more details see the websitesby Paul Webster and Herb Hagler, whichgive an excellent overview of many of thetechniques mentioned in this chapter. See:(http://www.hei.org/research/scientists/websterbio.htm).and (http://pathcuric1.swmed.edu/Research/haglerEMLab.htm>http://pathcuric1.swmed.edu/Res-earch/haglerEMLab.htm)

Acknowledgments

The need to cover such a broad area ofEM obliged me to seek the help of many

specialists for this chapter. I would firstlike to thank my colleagues from the an-nual EMBO-EM course for their support:Heinz Schwarz, Paul Webster, Herb Ha-gler, John Lucocq, Marek Cyrklaff, TerryMayhew, Ivan Raska, Peter Peters, PaulWalther and Kiyoteru Tokuyasu. A num-ber of these friends provided materialfor figures, acknowledged in the figurelegends. Collectively, they also helpedme enormously in the writing of thetext. I would also like to acknowledgethe help of Jacques Dubochet, Bill Earn-shaw, Helmut Gnagi, Kenny Goldie, AndyHoenger, Bruno Humbel, Eduard Kellen-berger, Kevin Leonard and Werner Villigerfor their input, and, in the case of BH,Jacques Dubochet, Andy Hoenger andBruno Humbel also for providing beau-tiful micrographs. Additional excellentimages were kindly provided by KazushiFujimoto, Shohei Yamashima, HermannFrank, Christoph Grunfelder, Jean-LucMurk, Peter Overath, Veronika Neubrand,Antonino Schepes, Anja Habermann andby Robert Ranner of Leica Microsystems,Vienna. Many thanks go also to HerbHagler, Paul Webster, Heinz Schwarz,Anja Haberman, and Mark Kuehnel fortheir great support in the preparationof the figures. Finally, a special thanksto Heinz Schwarz, to whom this chapteris dedicated, for giving me the pleasureof seeing his amazing collection of mi-crographs, the work of over 30 years,that cover almost all the techniques out-lined above.

Bibliography

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Adrian, M., Dubochet, J., Fuller, S. andHarris, R. (1998) Cryo-negative Staining.Micron. 29: 145–160.

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