microscopy - wordpress.com · 1772 but he was predominantly just a good manufacturer not inventor...

MICROSCOPY

Microorganisms: First to evolve -- last to be seen!

Resolving power of the eye

Relevant metric unitsmm = 10-3 meter

mm

nm

Resolution vs

Magnification

Earliest Microscopes• 1590 - Hans & Zacharias Janssen of

Middleburg, Holland manufactured the first compound microscopes

• 1660 - Marcello Malpighi (1628-1694), was one of the first great microscopists, considered the father embryology and early histology - observed capillaries in 1660 . Italian professor of medicine. Anatomist. First to observe bordered pits in wood sections. Gave first account of the development of the seed.

Early Microscopes (Hooke)

© J.Paul Robinson

The Royal Society of London founded in 1616 during the reign of King James I

•1665 - Robert Hooke (1635-1703)- book Micrographia, published in 1665, devised the compound microscope most famous microscopical observation was his study of

thin slices of cork. Named the term “Cell” - He wrote:

“. . . I could exceedingly plainly perceive it to be all perforated and porous. . . these pores, or cells, . . . were indeed the first

microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with

any Writer or Person, that had made any mention of them before this.” Robert Hooke

Oxford University

Overview of discovery

Earliest Microscopes•1673 - Antioni van Leeuwenhoek (1632-1723) Delft, Holland, worked as a draper (a fabric merchant); he is also known to have worked as a surveyor, a wine assayer, and as a minor

city official.

•Leeuwenhoek is incorrectly called "the inventor of the microscope" •Created a “simple” microscope that could magnify to about 275x, and

published drawings of microorganisms in 1683•Could reach magnifications of over 200x with simple ground lenses - however compound microscopes were mostly of poor quality and could only magnify up to 20-30 times. Hooke

claimed they were too difficult to use - his eyesight was poor.•Discovered bacteria, free-living and parasitic microscopic

protists, sperm cells, blood cells, microscopic nematodes •In 1673, Leeuwenhoek began writing letters to the Royal

Society of London - published in Philosophical Transactionsof the Royal Society

•In 1680 he was elected a full member of the Royal Society,joining Robert Hooke, Henry Oldenburg, Robert Boyle,

Christopher Wren

lens.exe

1670-1690

• Back: Italian compound microscopes - 1670

• Italian Compound microscopes• Back: 1670 (probably Campani)• This microscope was formerly at the

University of Bologna - it contains a field lens which was the first optical advance about 1660. Only opaque objects can be viewed.

• Front: Guiseppe Campani, Rome -1690 - Campani was the leading Italian telescope and microscope maker in the late `17th century - he probably invented the screw focusing mechanism shown on this scope - the slide holder in the base allows transparent and opaque objects to be viewed

Screwbarrel Microscope - 1720

• Made by Charles Culpeper

The issues between simple and compound microscope

• Simple microscopes could attain around 2 micron resolution, while the best compound microscopes were limited to around 5 microns because of chromatic aberration

• In the 1730s a barrister named Chester More Hallobserved that flint glass (newly made glass) dispersed colors much more than “crown glass” (older glass). He designed a system that used a concave lens next to a convex lens which could realign all the colors. This was the first achromatic lens.

Some Definitions• Absorption

– When light passes through an object the intensity is reduced depending upon the color absorbed. Thus the selective absorption of white light produces colored light.

• Refraction– Direction change of a ray of light passing from one transparent medium to

another with different optical density. A ray from less to more dense medium is bent perpendicular to the surface, with greater deviation for shorter wavelengths

• Diffraction– Light rays bend around edges - new wavefronts are generated at sharp

edges - the smaller the aperture the lower the diffraction• Dispersion

– Separation of light into its constituent wavelengths when entering a transparent medium - the change of refractive index with wavelength, such as the spectrum produced by a prism or a rainbow

Refraction

Light is “bent” and the resultant colors separate (dispersion).Red is least refracted, violet most refracted.

dispersion

Short wavelengths are “bent” more than long

wavelengths

Refraction

But it is really here!!

He sees the fish here….

Absorption

Control

No blue/green lightred filter

B & G absorbed

Light absorption

white light blue light red light green light

B & G absorbedR & G absorbed B & R absorbed

Absorption ChartColor in white light Color of light absorbed

red

bluegreen

magenta

cyan

yellow

blue

blue

blue

blue

greengreen

green

green

redred

redredblack

gray green bluepink

The light spectrumWavelength ---- Frequency

Blue light488 nm

short wavelengthhigh frequencyhigh energy (2 times the red)

Red light650 nm

long wavelengthlow frequency

low energy

Photon as a wave

packet of energy

θ1

θ2

θ3 θ4

Principles of light microscopy-- a single lens

Optical propertiesrefraction

magnification

resolution

distortion

Van Leeuwenhoek’s microscope

Limitations of a simple microscope

magnification

illumination

difficult to use

Robert Hooke’s microscope

Principles of a compound microscope

Ocular lens

Objective lens

Condensor lens

Calculating magnification

Hooke never reported seeing bacteria

Why?

Distortion is compounded

Compound ? (a neat idea)Simple Compound

Lenses and the bending of light

• Lens is a collection of prisms• Convex lens focuses light to the focal point.• Refractive index is a measure of how much

a subs slows the velocity of light.• Direction and magnitude is determined by

the RI of the two media forming the interface.

Nikon Labophot(Question 8)

Field lens(collects light)

Brightness control dial


Condenser(focuses light on specimen)


StageSlide holder

Stage X/Y-axis travel knobs


Focus knobs


Eyepiece(2nd level of

magnification)

Objectives(1st level of

magnification)

Nosepiece

Microscope Features

Illuminator: light source + collectorlens

Substage condenser: focuses light on specimen

Stage: specimen

Objective: 1st level of magnification(10X, 40X, 100X)

Nosepiece: to move objective

Eyepiece: 2nd level of magnification(10X)

ALWAYS MOVE MICROSCOPEBY GRASPING THE STAND AND

SUPPORTING THE BASE.

Compound Light Microscope

Magnification(Question 9)

Real image projectedby objective

Virtual image projectedby eyepiece

Total magnification= objective x eyepiece

Ex: 40X x 10X = 400X

Theoretical limit = 1000X> 1000X:

Image enlarged withno additional resolution

Magnification• Magnification: Degree of enlargement. i.e.

number of times the L, B, D are multiplied. • Limit of useful magnification set by

resolving power or limit of resolution.• Overall magnifying power obtained by

multiplying MP of objective with eye piece• Inclination of microscope head increases

magnification power of objective

• Optical tube length (160mm)/FL of objective

• Objective with FL 2 mm has a magnifying power of 80

• Objective lenses: – Low power FL 16mm– High power FL 4mm– oil immersion FL 2mm

RESOLUTION• The ability of the lens to differentiate between two small

objects.• Resolving power of a microscope is determined by

d=0.5/nsin• : Wavelength of light• Better resolution, shorter the wavelength. Therefore blue

range better -450-500nm• Limit if resolution of unaided eye=200 microns• LR of bright field microscope= 0.2 micron

Numerical Aperture• Ratio of diameter of lens to its Focal length FL (nsin )• is half the angle of cone of light entering the objective or

half the angle of aperture which is formed by 2 most divergent rays of light from the center of the object.

• Narrow cone of light has poor resolution.• Angle depends upon RI of the medium (n).• RI of air is 1.• Max NA =1 when working in air• resolution increased by adding oil

BRIGHTFIELD MICROSCOPE

Speed of light in vacuumSpeed of light in medium

What were the future advances in light microscopy?

1) Oil immersion lens

2) Fused lenses

3) Advanced optics

How do and oil immersion fused lenses improve resolution?

Refractive index =

Borosilicate glass = 1.52Air = 1.00Water = 1.33Immersion oil = 1.52

Fused lenses combine different types of glass-- compensate for ‘diffraction’

Objectives

ObjectiveType

SphericalAberration

ChromaticAberration

FieldCurvature

Achromat 1 Color 2 Colors No

Plan Achromat 1 Color 2 Colors Yes

Fluorite 2-3 Colors 2-3 Colors No

Plan Fluorite 3-4 Colors 2-4 Colors Yes

Plan Apochromat 3-4 Colors 4-5 Colors Yes

Numerical Aperture (N.A)

Air objective N.A ~ 0.95Oil objective N.A ~ 1.4

Water objective N.A ~ 1.25

ABILITY OF AN OBJECTIVE TO RENDER THE OUTLINE OF AN IMAGE CLEAR AND DISTINCT.

• Spherical aberration: Rays passing the edge of a lens not brought to same focus as those passing the center.

• Chromatic aberration: White light traversing a lens is separated into its component colours of different wavelegth, which are refracted to diff extent.

• Aberrations corrected by combining lenses of dispersive qualities

ObjectivesFunctions

1. Gather light from specimen2. Resolving power (reconstitute

the light from specimen into a clear image

3. Project a magnified real image into the body tube

Problems1. Chromatic aberrations2. Spherical aberration3. Curvature of field

The famous patent of 1758• George Bass was the

lens-maker that actually made the lenses, but he did not divulge the secret until over 20 years later to John Dollond who copied the idea in 1757 and patented the achromatic lens in 1758.

© J.Paul Robinson

Secondary Microscopes• George Adams Sr. made many microscopes from about 1740-

1772 but he was predominantly just a good manufacturer not inventor (in fact it is thought he was more than a copier!)

“New Improved Compound Microscope, George Adams, 1790Adams described this instrument in his “Essays on the Microscope” in

1787. The mechanism allowed freedom of movement. The specimen could be viewed in direct light or in light reflected from a large mirror.

© J.Paul Robinson

George Adams Toymaker to Kings

• This microscope made by George Adams, Mathematical Instrument maker to King George III around 1763, It was probably intended for the Prince of Wales, the future King George IV. The instrument is based on the design of the “Universal Double Microscope" (London Museum of Science)

Giovanni Battista Amici• In 1827 Giovanni Battista Amici, built high quality microscopes and

introduced the first matched achromatic microscope in 1827. He had previously (1813) designed “reflecting microscopes” using curved mirrors rather than lenses. He recognized the importance of coverslip thickness and developed the concept of “water immersion”

© J.Paul Robinson© J.Paul Robinson

Joseph Lister• In 1830, by Joseph Jackson Lister (father of Lord Joseph Lister) solved the

problem of Spherical Aberration - caused by light passing through different parts of the same lens. He solved it mathematically and published this in the Philosophical Transactions in 1830

© J.Paul Robinson

Joseph Lister

Pasteur - 1860

Louis Pasteur – his microscope was made in Paris by Nachet in about 1860 and was made of brass

Abbe & Zeiss • Ernst Abbe together with Carl Zeiss published a paper in 1877

defining the physical laws that determined resolving distance of an objective. Known as Abbe’s Law“minimum resolving distance (d) is related to the wavelength of light (lambda) divided by the Numeric Aperture, which is proportional to the angle of the light cone (theta)

formed by a point on the object, to the objective”.“The impetus for the emergence into the industrial age was given by Ernst

Abbe (appointed Associate Professor in 1870), who, while still in his early 30s, developed his theory of microscope image formation, which took into

consideration the familiar phenomenon of diffraction, and thus made the leap in microscope construction from trial and error to methodical design. He was given this commission by a university mechanic, Carl Zeiss, who had been

steadily perfecting the construction of optical equipment in his private workshops. Otto Schott, who received his doctorate at Jena in 1875, was the

third to enter into this alliance by founding, at Abbe’s urging, a "Laboratory for Glass Technology" in 1884, to produce the highly pure special lenses for

Zeiss’s microscopes and optical equipment. Humboldt’s pupil Matthias Jakob Schleiden, Professor of Botany and famous for his cell theory, encouraged --

and later benefited from -- this process, which was to prove exemplary in German economic history.”

http://www.uni-jena.de/History-lang-en.htmlAbbe

Abbe & Zeiss

• Abbe and Zeiss developed oil immersion systems by making oils that matched the refractive index of glass. Thus they were able to make the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing light microscopes to resolve two points distanced only 0.2 microns apart (the theoretical maximum resolution of visible light microscopes). Leitz was also making microscope at this time.

Zeiss student microscope 1880

Schott• Dr Otto Schott formulated glass lenses that color-corrected objectives

and produced the first “apochromatic” objectives in 1886.

Henri Hureau de Sénarmont (1808-1862)• Sénarmont was a professor of mineralogy and director of studies at the

École des Mines in Paris, especially distinguished for his research on polarization and his studies on the artificial formation of minerals. He also contributed to the Geological Survey of France by preparing geological maps and essays.

• Perhaps the most significant contribution made by de Sénarmont to optics was the polarized light retardation compensator bearing his name, which is still widely utilized today

Historical Figures

William Hyde Wollaston

William Hyde Wollaston (1766-1828) - Although formally trained as a physician, Wollaston studied and made advances in

many scientific fields, including chemistry, physics, botany, crystallography, optics, astronomy and mineralogy. He is

particularly noted for originating several inventions in optics, including the Wollaston prism that is fundamentally important to interferometry and differential interference (DIC) contrast

microscopy.

Georges Nomarski

• Georges Nomarski (1919-1997) - A Polish born physicist and optics theoretician, Georges Nomarski adopted France as his home after World War II. Nomarski is credited with numerous inventions and patents, including a major contribution to the well-known differential interference contrast (DIC) microscopy technique. Also referred to as Nomarski interference contrast (NIC), the method is widely used to study live biological specimens and unstained tissues.

Additional Information and Image at right from:ttp://micro.magnet.fsu.edu/optics/timeline/people/nomarski.html

Robert Day Allen

• Robert Day Allen (1927-1986) - Robert Day Allen was a renowned microscopist, a prominent researcher of cell motility processes, and a co-developer of video-enhanced contrast microscopy ((VEC)), which is a modification of the traditional form of differential interference contrast (DIC) microscopy. Along with Georges Nomarski and G. B. David, Allen assisted the Carl Zeiss Optical Company in developing a Nomarski differential interference microscope for transmitted light applications. In a hallmark paper published in Zeitschrift für wissenschaftliche Mikroskopie und mikroskopische Technik, Allen and his colleagues defined the basic principles of the DIC technique and the interpretation of images.

• Rebhun LI. Robert Day Allen (1927-1986): an appreciation. Cell Motil Cytoskeleton. 1986;6(3):249-55

More information at: (Image reproduced from below URL)ttp://micro.magnet.fsu.edu/optics/timeline/people/dayallen.html

Modern Microscopes

• Early 20th Century Professor Köhler developed the method of illumination still called “Köhler Illumination”

• Köhler recognized that using shorter wavelength light (UV) could improve resolution

Köhler

• Köhler illumination creates an evenly illuminated field of view while illuminating the specimen with a very wide cone of light

• Two conjugate image planes are formed– one contains an image of the specimen and the

other the filament from the light

Köhler Illumination

Specimen Field stopField iris

Conjugate planes for illuminating rays

Specimen Field stopField iris

Conjugate planes for image-forming rays

condenser eyepiece

retina

Magnification• An object can be focussed generally no closer than

250 mm from the eye (depending upon how old you are!)

• this is considered to be the normal viewing distance for 1x magnification

• Young people may be able to focus as close as 125 mm so they can magnify as much as 2x because the image covers a larger part of the retina - that is it is “magnified” at the place where the image is formed

Magnification1000mm

35 mm slide24x35 mm

M = 1000 mm35 mm

= 28

The projected image is 28 times larger than we would see it at 250 mm from our

eyes.If we used a 10x magnifier we would have

a magnification of 280x, but we would d h fi ld f i b f f 10

Some Principles

• Rule of thumb is not to exceed 1,000 times the NA of the objective

• Modern microscopes magnify both in the objective and the ocular and thus are called “compound microscopes” - Simple microscopes have only a single lens

Basic Microscopy

• Bright field illumination does not reveal differences in brightness between structural details - i.e. no contrast

• Structural details emerge via phase differences and by staining of components

• The edge effects (diffraction, refraction, reflection) produce contrast and detail

© J.Paul Robinson

Microscope Basics• Originally conformed to the

German DIN standard• Standard required the following

– real image formed at a tube length of 160mm

– the parfocal distance set to 45 mm– object to image distance set to 195

mm

• Currently we use the ISO standard

Focal lengthof objective= 45 mm

Mechanicaltube length= 160 mm

Object toImage Distance = 195 mm

The Conventional Microscope

Focal lengthof objective= 45 mm

Object toImage Distance = 195 mm

Mechanicaltube length= 160 mm

Modified from “Pawley “Handbook of Confocal Microscopy”, Plenum Press

Upright Scope

BrightfieldSource

Epi-illumination

Source

Image from Nikonpromotional materials

Inverted Microscope

BrightfieldSource

Epi-illumination

Source



Typical Inverted Microscope

Conventional Finite Optics

Sample being imaged

Intermediate Image

Telan Optics

Objective

Other optics

Ocular

45 mm

160 mm195 mm


Infinity Optics

Sample being imaged

Primary Image Plane

Objective

Other optics

Ocular

Other optics

Tube Lens

InfiniteImage

Distance

The main advantage of infinity corrected lens systems is the relative

insensitivity to additional optics within the tube length.

Secondly one can focus by moving the objective and not

the specimen (stage)


Images reproduced from:

http://micro.magnet.fsu.edu/

Advanced optics only improve contrast

Theoretical limit of resolution= 0.2 uM

Dark fieldmanipulates the path of light

Phase contrastand

Nomarskimanipulate phase of light

Dark field

Bright field

Phase contrast Nomarski

Phase contrast

• Unstained living organisms• Good contrast• High resolution• Objects dark grey against a light background.• Direct and diffracted rays from the objects which differ in their wave

phase, recombine so they interfere with each other. Hence reduce light intensity in the area of the image.

• Unstained bacteria have alternate areas of different refractive indices. These act as diffraction gratings. Differences are maximized by causing the direct and diffracted rays to pass through different thicknesses of glass in a phase plate.

• Phase plate retards diffracted rays one quarter of a wavelength with respect to direct rays.

• Condenser has an annular stop. Opaque disc with transparent ring.

Phase contrast

Phase contrast• Hollow cone of light produced.• When this cone passes thru a cell, some light rays are bent

due variations in density and RI and are retarded by 1/4 of wavelength

• Direct rays go through the phase ring in the phase plate which is present in the objective. Deviated rays miss the ring.

• Difference between the two is half the wavelength. If they recombine, they cancel each other.

• Useful for detecting bacterial components.• Also used in studying eukaryotes.

Darkground Microscope

• Bacteria brightly lit against a dark background.• Can observe living unstained organisms• Specimen illuminated by rays of light so oblique that unless scattered

by objects, they do not enter the objective.• Darkground condenser- The special condenser incorporates concentric

reflecting mirrors. A central one prevents light rays from passing directly up and passes them onto peripheral mirrors. The latter reflects rays Optical system enhances the contrast of unstained bodies.

• inwards onto specimen at a very oblique angle.• High intensity lamp• Funnel stop which reduces the numerical aperture to less than 1. It

consists of a funnel shaped piece of metal or palstic and is fitted into the objective.

• Particularly useful in observing smaller spirochetes

Schematics of the ray paths in a dark-field contrastmicroscope. Rays fall into the objective lens only if theincoming light is scattered at the specimen in the focal plane

Dark-field contrast

Immunofluorescenceand

Confocal Microscopy

Part I: Immunofluorescence

Principle of Fluorescence Microcopy


Exciter filter


Exciter filter Barrier filter

In fluorescence microscopy, electrons in labeling agent molecules(here for simplicity drawn as single atoms with electron shells) ofthe fluorescence species are excited by light with shortwavelength to higher energetic molecular orbitals. The excitedelectrons loose their energy by emission of fluorescence light withlonger wavelength compared to the excitation radiation. Theeminent light is used for image production in the microscope.

Fluorescence microscopy

Fluorescence microscope

• Light source is UV mercury vapor lamp• UV light is filtered to select excitation light

to pass through• Excitation light is reflected by a dichroic

mirror to strike on the specimen • Emission light passes through the dichroic

mirror


• Barrier filter blocks the excitation light amid the light path to visualization

• Fluorescent labels are visulized against a dark background


• The combination of exciter filter, dichroic mirror and barrier filter should be selected according to the fluorochrome label

• The 3 components are usually built into a single module called the filter block

Epi-fluorescence microscope

Transmitted light flurorescence microscope

The basic principle of immunofluorescence

• To use a fluorescent compound (usually fluorescein) to detect the binding of antigenand antibody.

• The Ab is labeled with the fluorescent compound and its presence is detected using a fluorescence microscope.

• Under a fluorescence microscope, fluorescein appears bright green wherever the binding occurred.

Exciter filter

The basic principle of immunofluorescence

• To use a fluorescent compound (usually fluorescein) to detect the binding of antigenand antibody

• The Ab is labelled with the fluorescent compound

• Under a fluorescence microscope, fluorescein appears bright green wherever the binding occurs

Using the fluorescence microscope

• Select the correct filter block for the fluorescent compound

• Fluorescence fades quickly under UV light; try to limit the time of exposure to UV as much as possible

• Use high speed films for photography

Direct Immunofluorescence

• The aim is to identify the presence and location of an antigen by the use of a fluorescent labelled specific antibody

One step Direct Immunofluorescence

Two step Direct Immunofluorescence

Medical applications of direct IF

• Renal diseases for evidence of immune deposition

• Skin diseases for evidence of immune deposition

• Detection of specific antigens, especially those of infective organisms

Application in renal diseases

IgG

• A section of kidney is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away

• The presence of fluorescence in the glomeruli indicates that IgG was deposited prior to the biopsy

• IgG is deposited in granular clumps along the capillary walls, enabling a diagnosis of membranous glomerulonephritis in this case

Direct Fluorescent Antibody Test for the Presence of Immunoglobulin Deposits in Skin

IgG

• A section of skin is placed on a slide; a fluorescein-labeled antiglobulin (specific for IgG, in this case) is added, then rinsed away

• The presence of fluorescence in the upper layers of the epithelium indicates that IgG was deposited in this skin (prior to the biopsy)

• The presence of immunoglobulins deposited around keratinocytes is consistent with a diagnosis of pemphigus

Double labelling

Lymphoid tissue:the two Ig light chains are separately labelled.

Indirect Immunofluorescence


• The aim is to identify the presence of antigen specific antibodies in serum. The method is also be used to compare concentration of the antibodies in sera.


• A known antigen is placed on a slide; the patient's serum is added, then rinsed away.

• A fluorescein-labeled antiglobulin is added, then rinsed away.

• The presence of fluorescence over the antigen indicates the presence of antibodiesto this antigen in the patient.

Diagnosis of Bacterial Diseases

• Clostridial diseases (direct) • Brucella canis (indirect) • Afipia catei, cat scratch disease (indirect) • Borrelia burgdorferi (indirect) • Coxiella burnetii, Q Fever (indirect) • Rickettsia rickettsiae, Rocky Mountain

Spotted Fever (indirect)

Diagnosis of Viral Diseases

• rabies virus (direct)• bovine immunodeficiency-like virus (indirect)• canine coronavirus (indirect)• canine distemper (indirect)• feline infectious peritonitis (corona-) virus

(direct)• porcine respiratory and reproductive syndrome

(indirect)

Diagnosis of Protozoal Diseases

• Babesia species (indirect)• Ehrlichia species (indirect)• Toxoplasma gondii (indirect)• Trypanosoma cruzi (indirect)• Cryptosporidia/Giardia (direct)• Encephalitozoon cuniculi (indirect)• Neosporum caninum (direct, indirect)

Some examples


Indirect Fluorescent Antibody Test for Antibodies to Toxoplasma gondii


• Toxoplasma organisms are killed and placed on the slide; the patient’s serum is added, then washed away.

• A fluorescein-labeled antiglobulin is added, then washed away.

• The presence of the green fluorescence outlining the T. gondii organisms indicates the presence of antibodies in the patient's serum.

Immune-Mediated Disorders

• antinuclear antibody (ANA) test (for diagnosis of systemic lupus erythematosus)

• Direct fluorescent antibody test for deposition of Abs in tissues, e.g. kidney, skin

Indirect Fluorescent Antibody Test for Antinuclear Antibodies


• Cells from a cultured cell line are placed on a slide; the patient's serum is added, then rinsed away.

• A fluorescein-labeled antiglobulin is added, then rinsed away.

• The presence of fluorescence in the nucleus of these cells indicates the presence of antibodies to nuclear antigens in the patient.

Advantage over Immunoperoxidase

• Technically easier (fewer steps)• More sensitive results

Drawbacks

• Microscope is more costly• Frozen sections preferred• Preparations need refrigeration• Preparations cannot be kept for too long• Quick fading of fluorescence under

illumination (bleaching effect)

Part II: Confocal microscopy

Principles of confocal microscopy

• a focused laser beam serves as a high intensity point source

• light reflected or fluorescence emitted by the specimen is allowed to pass through a pinhole that filters light coming from outside (above and below) of the focal plane

Principles of confocal microscopy

• a sensitive detector (photomultipler) behind a pinhole to measure the intensity of light

• the laser beam, the pinhole and detector scan through the specimen to build up an image on a monitor

The confocalconcept

Modes of scanning

• Mechanical scanning stage• Beam scanning (by means of mirror)• Combined stage and beam scanning• Slit may be used instead of a pinhole

– Shortens time for scanning an area– Direct vision of real color image made possible– At the expense of a lower resolution

Use of confocal microscope

• Performs optical sectioning of thick samples• Three dimensional image reconstruction• Detects very weak fluorescent signals• Selective photobleaching• Cell ablation

Image modalities

• Autofluorescence• Single, double or treble fluorescent

labeling:– immunofluorescence, in-situ hybridization

• Image formed by reflectance intensified with metallic coating e.g. AgNOR, immunogold labeling

Application in biomedical science

• Growth of small organisms, cells, embryos• Movement of intracellular structures• Change in membrane permeability• 3 dimensional reconstruction• Image analysis

BIO-RAD MRC-1000

• beam scanning LSM• Zeiss "Axioskop" upright microscope for ordinary

bright field transmitted light and epi-fluorescence• argon/krypton laser, 15 mW, wavelengths at 488

nm (e.g. FITC), 568 and 647 nm• reflectance mode• fluorescence mode:

– simultaneous recording of 2 channels, ie. For double labels

BIO-RAD MRC-1000

• upgradable to 3 simultaneous channels in fluorescence mode

• computer-controlled stage motor for vertical motion at 0.5µm per step

• performs frame scan, line scan, vertical scan• maximum image size 1024 x 1024 pixels• scanning speed at maximum of 16 frames/s

at 768 x 30 pixels, and 1 frame/s at 512 lines

BIO-RAD MRC-1000

• beam park function for photobleaching, ablation

• computer programs for morphometric analysis, 3-D visualization

IgG

IgG

Confocal

Confocal microscope gives a clearer image andcleaner background overconventional fluorescent

microscope

Microphotographyfor weak signalsis much easierwith a confocal

microscope

Microphotographyfor weak signalsis much easierwith a confocal

microscopeExcellent for picking up weak signals

How do electron microscopes yield higher resolution?

Resolution ~ 0.2 nM (0.0002 uM)- light microscope ~ 0.2 uM- eye ~ 0.2 mm

Electrons have very small wavelength

Wavelength of visible light ~ 550 nm… of electrons ~ 0.005 nm

What are the two types of EM?

Scanning vs Transmission

False coloration common

.

Optical principles of (A) confocal laser scanning microscopy (CLSM) and (B) multi-photon excitation microscopy (MPEM).

Method of use microscope

• Eye piece in level with observer’s eye.• Check posture• Objectives and Eye pieces should be free of dirt.• Use only Benzol or Xylol to clean lenses.• View specimen in low power, then move to next objective.• Use fine focus only when image is in focus.• Adjust eye pieces so single field is seen.• If can’t focus, check for dirt or oil on lenses.• Slide has a film of oil?• Slide upside down. • Coverslip too thick

microscopy - wordpress.com · 1772 but he was predominantly just a good manufacturer not inventor...

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