analytical cellular pathology 34 (2011) 5–18 ios press optical...

Analytical Cellular Pathology 34 (2011) 5–18DOI 10.3233/ACP-2011-0006IOS Press

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Optical and digital microscopic imagingtechniques and applications in pathology

Xiaodong Chena,c, Bin Zhengb and Hong Liuc,∗aCollege of Precision Instruments, Tianjing University, Tianjing, ChinabDepartment of Radiology, University of Pittsburgh, Pittsburgh, PA, USAcCenter for Bioengineering and School of Electrical and Computer Engineering,University of Oklahoma, Norman, OK, USA

Abstract. The conventional optical microscope has been the primary tool in assisting pathological examinations. The mod-ern digital pathology combines the power of microscopy, electronic detection, and computerized analysis. It enables cellular-,molecular-, and genetic-imaging at high efficiency and accuracy to facilitate clinical screening and diagnosis. This paper firstreviews the fundamental concepts of microscopic imaging and introduces the technical features and associated clinical applica-tions of optical microscopes, electron microscopes, scanning tunnel microscopes, and fluorescence microscopes. The interfaceof microscopy with digital image acquisition methods is discussed. The recent developments and future perspectives of contem-porary microscopic imaging techniques such as three-dimensional and in vivo imaging are analyzed for their clinical potentials.

1. History of microscope development

More than 2000 years ago, around one century B.C.,people already had discovered that one could view anenlarged image of a small object by using a transparent“lens” with a spherical shape. Although a single con-vex lens can magnify an object by more than ten times,it is quite insufficient for people clearly to observethe details of many small objectives [1]. Near the endof 16th century, Dutch eyeglass dealer, Janssen, andhis son inserted several lenses into a cylinder and foundthat an object was enlarged significantly if viewedthrough this assembled cylinder. This was the first pro-totype of a modern microscope and telescope. Based onthis accidental discovery, Janssen designed and assem-bled the first compound microscope that includedtwo convex lenses. The basic concept of a compoundmicroscope is that an object can be enlarged sequen-tially by two convex lenses.

∗Corresponding author: E-mail: [email protected].

While observing the soft-wood specimen usinga microscope in 1665, the British scientist, RobertHooke, surprisingly discovered that the specimendepicted a series of unique “elements.” Hooke namedthem “cells.” Years later, a new microscope designedand assembled by a Dutch scientist, Anthony vanLeeuwenhoek, provided much higher magnificationpower, which enabled people to observe substantiallymore details of cells. Although it exceeded all prece-dent microscopes, it was not another compound micro-scope and actually used only one convex lens.However, due to Leeuwenhoek’s excellent manufac-turing skill, this specially manufactured single convexlens achieved more than 300 times of magnificationpower.

In the following two centuries, the magnificationpower, as well as the image quality of the compoundmicroscope, was improved substantially. This occur-red, in particular, after the discovery and use of the newlens assemblies were able to eliminate or minimize thechromatic and other optical aberrations. Compared to

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6 X. Chen et al. / Optical and digital microscopic imaging techniques and applications in pathology

the microscopes developed in 19th century, the con-ventional optical microscopes used to date have nosubstantial difference or improvement, because theoptical microscopes have reached their maximum lim-itation in the spatial resolution.

Due to the large range or fluctuation of optical wave-length, it is impossible for any optical instrumentsto create a perfect image of a natural object, even ifall shape defects in manufacturing optical lens wereeliminated. Due to the unavoidable diffraction of opti-cal wave passing through the microscopic lens, anypoint depicted on an object is no longer the point inthe image plane but a diffraction spot. If two diffrac-tion spots are too close together, the two points arenot distinguishable. Under such circumstance, increas-ing magnification power of the microscopic objectivelenses cannot further increase the spatial resolution ofthe microscopes. For the microscopes using the lightsources within the range of visible optical wavelength,their spatial resolution is limited to 0.2 �m. Thus, anystructures smaller than 0.2 �m cannot be distinguishedusing this type of microscope.

One approach to increasing spatial resolution ofmicroscopes is to reduce optical wavelength or useelectron beam to replace visible light source. Accord-ing to the de Broglie theory of matter wave, movingelectrons is similar in nature to optical wave fluctu-ation. The faster that electrons move, the shorter the“wavelength” of the released energy. If the electronscan be accelerated sufficiently and the released energycan be converged, the moving electron beam also canenlarge the objects. Based on this concept, Germanengineers, Max Knoll and Ernst Ruska, assembled thefirst transmission electron microscope (TEM) in theworld in 1938 [2]. British engineer, Charles Oatley,manufactured the first scanning electron microscope(SEM) in 1952 [3]. Electron microscopy was one ofthe most important inventions in 20th century. Becauseelectrons can be accelerated to very high speed, thespatial resolution of electron microscopes reaches upto 0.3 nm. As a result, many invisible materials (i.e.,virus) under the visible light become “visible” underthe electron microscope.

In 1983, two scientists in IBM laboratory, GerdBinning and Heinrich Rohrer, invented the scanningtunnel electron microscope (STM) [4]. This advancedmicroscope was built using totally different conceptsthan those used by the conventional microscopes. STMworks based on the so called “tunnel effect”. STM hasno lens; rather, it uses a probe. Once the voltage is

added between the probe and the observed object, thetunnel effect occurs if the distance between the probeand the surface of the observed objects is sufficientlysmall (i.e., measured in nanometers). When the elec-trons pass through the tiny space between the probe andthe object, weak electronic current is generated. If thedistance between the probe and the object varies, thestrength of the current varies as well. Hence, the three-dimensional shape of the object can be detected as onemeasures the electronic current change. The spatial res-olution of the STM can reach the level of a single atom.

2. The basic concept of microscopic imaging

Although many different types of microscopes havebeen developed, the basic imaging concept and struc-tures can be simply illustrated in Fig. 1. The opticalsystem of a microscope mainly includes an objectivelens and eyepieces. The purpose of an objective lens isto magnify an object so that it can be clearly observedby the user. During the observation, the specimen isplaced near the focal plane of the objective lens in theobject space, and a magnified real image of specimenis first created on the intermediate plane. The interme-diate plane is located on the focal plane of the eyepiece,thus the eyepiece is working as a magnifier to furthermagnify the image projected on the intermediate imageplane. Finally, a magnified, virtual, inverted image isprovided for the observer.

For a well designed microscope, the spatial res-olution is mainly determined by the objective lens.Although an eyepiece can also magnify the image,it cannot improve the resolving power of the micro-scopes. The spatial resolution of an optical microscopeis given by the Rayleigh equation as follows [5]:

�r0 = 0.62λ/n sin α (1)

where �r0 is the minimum resolvable distance, λ

is the wavelength of the light source; n is the refrac-tive index between the lens and the object, � is thehalf-angle aperture – the half inclination of the lens tothe objective points, and n × sin(�) is the numericalaperture (NA) of the objective lens.

In the case of digital microscopic systems, the imagemagnified by the objective lens is directly acquired (orthrough a relay lens) by electronic detectors such asCCD or CMOS detectors [6, 7]. The electronic detec-tors are selected so that their pixel pitch is smaller

X. Chen et al. / Optical and digital microscopic imaging techniques and applications in pathology 7

Fig. 1. The optical principle of microscope imaging.

than the magnified minimum resolvable distance deter-mined by the objective lens. So that the resolvingpower of the microscope is not degraded by the digitaldetectors.

Based on the above equation, and considering thefollowing practical limitations, (1) the use of visiblelight with the wavelength between 390 nm and 760 nm,(2) the maximally reachable aperture with the half-angle of 70–75 degree, and (3) the requirement ofusing immersion methods with water or oil to increaseof refractive index, the resolution of a conventionaloptical microscope cannot exceed 200 nm.

Since the wavelength of electronic wave (beam) ismuch shorter than the visible light waves in severalorders of magnitude, the resolution of a microscopeusing electronic beam in theory can reach approxi-mately 0.3 nm. For this reason, electron microscopy[8, 9] is developed based on the principles of elec-tronic optics, which can image the fine structure ofobjects at very high magnification power by using theelectron beam and electron lens instead of optical lens.The transmission electron microscope (TEM) is theprimary representation of the electron microscopes.The TEM is so named because the electron beam firstpenetrates the sample and then is magnified by the elec-tronic imaging lens to produce the images. Its opticpath is similar to the conventional optical microscope(as shown in Fig. 2). After passing through convergentlens, the parallel electron beam reaches the sample.Once the electron beam passes through the sample, itcarries the information related to the sample charac-teristics. An electronic image is first generated once

Lamp

Optical lens

Optical lens

Optical lens

Ocular

Eye

Illumination

Condenser lens

Specimen

Objective lens

First image

Projector lens

Final image

Electrons

Electro-magnetic lens



EyeFluorescent screen

Fig. 2. Comparison of the optical microscopic and electron micro-scopic systems.

the electron beam passes though the objective lens andcontrast aperture. After the electronic image is magni-fied again by the intermediate lens and projection lens,the final electron image is displayed in the electronicmonitor screen. The contrast of the electron image isdetermined by the scattering level of electron beamonce it interacts with the atoms in the sample. Theelectron beam also scatters less in the thinner samplesor some parts with lower density. Thus, more electronspass through lens aperture and result in the brighterimage. On the other hand, thicker samples or denserparts appear darker in the image. If the sample is too


thick or too dense, the image contrast is decreasedsubstantially.

3. The microscopes used in biomedical fields

Although a large number of different types of micro-scopes have been developed and applied in differentapplications, the microscopes actually can be dividedinto three categories: (1) optical microscopes, (2) elec-tron microscopes, and (3) scanning tunnel electronmicroscopes based on the microscope developmenthistory.

3.1. Optical microscopes

The simplified optical wave path of a conventionaloptical microscope is illustrated in Fig. 3. The mod-ern optical microscope is able to magnify an objectby 1500 times with the 0.2 �m limit in spatial reso-lution. The optical microscopes can be divided intomany different types using a variety of criteria. Forexample, based on a lighting method, there are thetransmission and reflection types of microscopes. Ina transmission microscope, the light passes throughtransparent objects. In a refection microscope, the lightsource installed on the top of the microscopic lens illu-minates the non-transparent objects, and the reflected

Light source

Eyepieces

Objective lens

Specimen

Condenser

Fig. 3. Illustration of optical path of the microscope.

light is collected by the lens. The microscopes can alsobe differentiated based on the observation methods,including bright field microscopes, dark field micro-scopes, phase difference microscopes, polarized lightmicroscopes, interference microscopes, and fluores-cent microscopes [5, 10–13]. Each microscope canuse either the transmission or reflection approach.The bright field microscopes are the most popularand widely used of all microscopes. Using this typeof microscope, the transmission (or absorption) ratioand reflection ratio of some observed objects varyaccording to the change of working environments. Theamplitude of these objects varies with the change inlighting intensity. The colorless transparent objectsare visible only when the phase of illuminated lightchanges. Because the bright field microscopes cannotchange light phase, the colorless transparent specimensare invisible when using this type of microscope.

3.2. Electron microscopes

The resolution of the electron microscopes typicallyis represented by the small distance of two adjacentand distinguishable points. In the 1970s, transmissionelectron microscopes were able to reach the reso-lution of 0.3 nm, while the resolution limit of thehuman eye is around 0.1 mm. Compared to the opti-cal microscopes that have the maximum magnificationpower of a few thousand times, the modern electronmicroscopes can increase the maximum magnificationpower to more than 300 million times. Using electronmicroscopes, one can directly observe some biolog-ical structures and/or the atomic structures of cells.Despite having superior spatial resolution over theoptical microscopes, electron microscopes must workunder the vacuum environment. Thus, they cannot beused to observe living biology samples. In addition, theelectron beam can damage the illuminated specimens.Other issues (i.e., the optimal control of brightness ofelectron bean gun and the improvement of electron lensquality) still need investigations [14].

3.3. Scanning tunnel microscopes

The scanning tunnel microscope (STM) is alsonamed “tunnel scanning microscope”. This instrumentdetects the surface structure of the objects based on thetunnel effect of the quantum mechanics. STM appliesa very thinning tip (in an atom unit of its head) to


detect the sample surface. As the tip is very close tothe sample surface (<1 nm), the atoms in the tip headoverlap with the electronic cloud of electrons in thesample surface. If a biasing voltage is applied betweenthe tip and the samples, electrons will pass through thebarrier between the tip and sample to generate the tun-nel current in the order of nano-amperes (10−9 A). Bycontrolling the distance between the tip and the sam-ple surface and accurately moving the tip along thesample surface in the three-dimension, the detector canrecord the data related to the surface morphology, elec-tronic surface states, and other relevant sample surfaceinformation [15]. The STM has the amazing ability toachieve the spatial resolution of less than 0.1 nm in thehorizontal direction and less than 0.001 nm in verticaldirection. Generally, when objects are in the solid state,the distance between atoms typically is from 0.001 nmto 0.1 nm. Because of this, the STM enables scientiststo locate the single atom and observe the status of theatoms and molecules in the conductive material andsurface structure. Hence, the STM generates substan-tially higher spatial resolution than other similar atomicmicroscopes. On the other hand, STMs can use the tipof the probe needle to manipulate accurately the atomsunder low temperature environment. This enables theSTM to be used as both a measurement and a processtool in the nanometer technology [16–18]. One uniquecharacteristic of STM is that, for the first time, onecan observe the permutation status of the atoms in theobject surface and the related physical chemistry char-acteristics of the electrons in the surface. Due to thewidth and scope of its perspectives in the research andapplication fields related to the surface, material, andlife sciences, the invention of the STM was consideredby the international scientific community as one of theten most important achievements of the science andtechnology in 1980s.

In summary, increasing microscope resolution hasbeen an actively pursued goal in the microscopic imag-ing field. Using these three types of microscopes, onemay observe the biological cells and microorganismsusing an optical microscope, observe virus using anelectron microscope, and detect or visualize the atomsusing a scanning tunneling microscope.

3.4. Fluorescence microscopes

Although the fluorescence microscope can be char-acterized as a branch of the optical microscopes, it has

many unique image generation and application charac-teristics in the biomedical imaging research fields. Thissection discusses this in more detail. The fluorescencemicroscope [19] uses short-wave light to illuminate theexamined specimen and make certain objects insidethe specimen emit the fluorescent light. Based onthe received fluorescent light, one is able to observe theshape and location of the target objects. Comparing thedifference between the fluorescence and conventionaloptical microscopes, one of the major differences isthat the fluorescence microscope uses the high-voltagemercury lamp to provide all band light illumination,using a projection method in which the light is pro-jected to the specimen. Due to the typically weaklyreflected fluorescent light, the microscope must havea large numerical aperture to allow the objects to beobserved. As a result, the numerical aperture of thefluorescent microscopes is larger than conventionaloptical microscopes. The fluorescent microscopes alsohave a special filtering system, including the assemblyof the illumination filters and cut-off filters. Specif-ically, the fluorescent microscope has the followingunique characteristics or requirements:

1) It requires a light source that has sufficient powerto emit fluorescent light;

2) It is equipped with a set of optical filters that fitthe requirement of stimulating different objectsto emit different fluorescent light. By selectingthe suitable wavelength of the emitting light, thewavelength of the emitted light can coincide withthat of the absorption light, resulting in the max-imum fluorescent light output;

3) To acquire the weak fluorescent images, it isequipped with a set of cut-off light filters thatallow only the selected fluorescent light to passthrough the imaging system and block the othertypes of scattering light to increase the signal-to-noise ratio of the system; and

4) The system of optical magnification needs to fitthe characteristic of the fluorescent light in ordereventually to obtain the fluorescent images withthe highest spatial resolution.

Due to these requirements, a modern fluorescentmicroscope typically is assembled based on the struc-ture of a duplex optical microscope. The equippedfluorescent device includes fluorescent light source,emission light path, stimulation/emission light filters,and the other components.


1) The fluorescent light source: It provides the lightsource that emits the light within the specificrange of the wavelength or energy, which guar-antees that the examined specimen acquiressufficient stimulation and generates strong flu-orescent light. The fluorescent microscope typi-cally uses the mercury lamp as the light sourcethat is able to provide the exciting light withcontinuous wavelength. The mercury lamp hasstronger energy in several commonly used lightwavelengths (including 365 nm, 405 nm, 550 nm,and 600 nm).

2) An exciting light path: It serves as a fluorescentlighting device that contains a group of condenserlens and light focusing adjustment device. In thelight path, the adjustable light and field apertures,(Neutral Density) ND filters, light splitter, andprocessing lens are installed.

3) Light filters: These are a set of the optical filter-ing components. Each has a certain wavelengthwidth that selectively allows stimulated/emittedlight pass through the optical system of themicroscope.

Similar to the other conventional optical micro-scopes, the fluorescence microscopes can be dividedinto two categories based on their optical path [20]:transmission fluorescence microscopy (TFM) and epi-fluorescence microscopy. In TFM, the light passesthrough the condenser to excite the specimens toemit the fluorescence light. The TFM often uses thedark-field condenser that is able to adjust a reflec-tor so that the light can illuminate the specimenfrom different directions. Compared to the old stylefluorescent microscopes, the TFM has a number ofadvantages, which include producing strong fluores-cence light when the specimen is observed in the lowmagnification. Disadvantages include the fact that asmagnification levels increase, the fluorescence lightweakens. Therefore, TFM is better used for observ-ing larger specimens. As shown in Fig. 4, the light isemitted from one side of the specimen and the fluores-cent light is collected by the lens in the other side ofthe specimen.

The recently developed epifluorescence microscopyis a new type of fluorescent microscopy [21]. Thedifference between the epifluorescence and the trans-mission fluorescence microscopy is that the objectivelens in epifluorescence microscope serves first asa well-corrected condenser and then as the image-

LightSource

Detector

Filter

ObjectiveFluorescence

Specimen

Condenser

Reflector

Fig. 4. Illustration of optical path of a transmission fluorescencemicroscopy.

forming light gatherer. The illuminator directs lightonto the specimen by first passing the light through themicroscope objective lens on the way toward the spec-imen and then using the same objective lens to capturethe emitted light. The dichroic beam splitter is placedbetween the optical path titled at 45◦, and the reflectedexcitation light passes through the objective lens andeventually reaches the specimen. The fluorescent lightemitted by the specimen, as well as the reflected excit-ing or scattering lights, passes through the objectivelens simultaneously and arrives at the dichroic separa-tor (mirror) that separates the fluorescent light and theoriginal exciting light. The remaining scattering lightis absorbed further by the cut-off filter. The advantagesof the epifluorescence microscope include the uniformillumination, sharp image, and the strong fluorescentlight as the increase of the magnification level.

The optical path of an epifluorescence microscopeis illustrated in Fig. 5. After the reflection from thedichroic mirror, the excitation light converges at thespecimen. The emitted fluorescent light from the spec-imen first passes through the objective lens and thedichroic mirror, and then is collected by detection sen-sor (probes). The key component of an epifluorescencemicroscope is the dichroic beam splitter. It includes amultilayer optical interference filter that is installed


LightSource

Detector

Specimen

Fluorescence

Objective

Objective

DichroicMirror

Filter

Fig. 5. Illustration of the optical path of a typical epifluorescencemicroscopy.

with a titled angle of 45◦ along the optical path. It isable selectively to reflect or transmit the light only incertain wavelength range.

Although the transmission fluorescence micro-scopes can yield images with sufficiently high contrastin low magnification, the image is often relatively darkcompared to the use of the epifluorescence microscope.Thus, the epifluorescence microscope is more popularin clinical practice to date. In addition, compared tothe transmission fluorescence microscope, the epiflu-orescence microscope has following characteristics.

1) The microscope objective lens also serves as alight condenser. Thus, the alignment issue of thecondenser is avoided. In particular, the fluores-cent light intensity of the image increases as theincrease of the numerical aperture (NA) of theobject.

2) Since excitation light does not pass through slidesin epifluorescence microscopy, it reduces thelight loss. Meanwhile, the excitation and fluo-rescent light travel in the opposite direction tothe objective lens. Two light beams separate witheach other without interference. Some scatteredexcitation light may reach the object, and theyare mostly reflected by the dichroic mirror to

the light source. Thus, a very thin filter typi-cally is installed between the objective lens andthe detector (probe) to absorb a small fractionof remaining scattering light passing through theobjective lens.

3) Unlike the use of transmission-type of excitation,in which most of fluorescence light creates in thebottom section of the specimen and must transmitthrough the specimen slice producing unavoid-able scatter light, in epifluorescence microscope,the light source and the observation plane locatein the same plane. Therefore, there is no loss ofthe fluorescence intensity and no effect on imagequality. In particular, it has unique advantageswhen it is used to examine the thick specimenslices with bacteria and tissue culture.

4) Since the ultraviolet (UV) light needs to passthrough the objective lens, the transmission coef-ficient of the objective lens must be considered.As a result, a series of special objective lenses thatcan transmit UV light have been designed andmanufactured. However, such a special objectivelens is not required in the transmission fluores-cence microscope.

4. The application of microscopesin pathological imaging

The invention of the microscope has significantlypromoted the development of biology and medicine.It demonstrates the mystery of the microscopic worldand reveals the causes of disease from the microscopicview. By acquiring the microscopic and pathologicimages of the pathogenic microorganisms, medicalresearchers have established a series of new aca-demic subjects, including bacteriology, immunology,virology, cell pathology, and others. In particular, theinvention of electron microscopes has raised the obser-vation of the traditional pathology from the cellularlevel to the sub-cellular level. As a result, one can-not practice modern medicine without microscopes.New microscopes such as the confocal laser scanningmicroscope (CLSM) [22], atomic force microscope(AFM), and scanning tunneling microscopy (STM)further improve the resolution of pathology imaging[23]. In this section, we briefly discuss the applicationof microscopes in assisting the clinicians in biomedicalengineering, particularly in diagnosing pathologicalimages.


4.1. Optical microscopes

Optical microscopes have been used widely in thepathology related clinical laboratories to diagnose avariety of diseases based on the examination of thebody fluid change, invaded virus, and the variationof atomic structures [24]. Such diagnostic informationand reports provide the clinicians with valuable refer-ences to select and implement the optimal treatmentplans for the patients and monitor their efficacy. Ingenetic engineering and microscopic surgery, micro-scopes are essential tools for clinicians. Althoughoptical microscopes are still considered to be veryimportant tools in the clinical facilities, the develop-ment of the originally optical microscopes is quitemature to date; in particular, the optical characteris-tics of the microscopes have reached perfect degreewithout much space left to make further improve-ment. Therefore, the current development of the opticalmicroscopes focuses on finding new application fields,making the systems simple and easy to use, as well asmaking one system for the multiple applications.

Since the biological specimen is typically one kindof muddy medium, it can generate the strong lightscattering and absorption. As a result, the light (oroptical wave) cannot penetrate deeply into the inte-rior of the biological structures; thus, it is unable toextract a clear image of the biological structures. Withthe rapid advance of photonics, new imaging methodsbased on photonics have been developed, including thelaser confocal scanning microscope and the near-fieldoptical scanning microscop [25, 26]. First, the laserconfocal scanning microscope is the combination oflaser source and the confocal microscope. Specifically,it is a system that uses both the confocal concept of thetraditional optical microscopy and the laser as the lightsource. It then uses the computer to process and analyzethe acquired digital images of the observed specimens.By continuously scanning the multiple layers of theliving cells or tissue slices, the laser confocal scanningmicroscope can acquire the complete 3D images ofeach structural layer of a single cell or local structureof a group of cells. Based on the near-field probingtheory, the near-field optical scanning microscopy is anew technology developed by using the optical scan-ning probe needles. It breaks through the limitation ofoptical diffraction and is able to reach the spatial res-olution in the range from 10 nm to 200 nm. By furthercombining with the related optical spectroscopy, usingthe near-field optical scanning microscopy creates a

new approach to detecting and examining spectrumimages of small biological specimen beyond nanome-ter range. As a result, researchers are able to analyzethe single biological atom to date.

4.2. Electron microscopes

There are number of differences between electronicand optical microscopes when applied to pathologicalimages. When using optical microscopes to observepathological specimens, the observer routinely needsto adjust the micro-focusing system of the microscopeand move the specimen to achieve the optimal obser-vation of the tissue structure of the specimen. Usingoptical microscopes also generates less eye fatigue dueto small light simulation to the eyes. However, themagnification power of the optical microscope is muchlower than that of the electronic microscope. In addi-tion to the higher magnification power, an electronicmicroscope can display more detailed tissue structureson the monitor (screen). It also may be able to conductsecondary magnification of the specimen displayed onthe screen. Thus, the electronic microscope plays animportant role in studying human organ tissue struc-tures and the other sub-nanometer structures.

Due to the short wavelength of the electron beams,the electrical microscopes break through the limita-tion of the spatial resolution using optical microscopes,which opened a new door for the human eyes to observethe small structures at the molecule and/or atomic level.For example, using electronic microscopes to observecells, researchers clearly have confirmed the existenceof a cell membrane that consists of three thin layerswith equal thickness but different density. Two exter-nal layers have higher density than the middle (interior)layer. When using electronic microscopes to observeunmyelinated nerve fiber, it was found that the nervemembrane cell of the fiber contained multiple shafts(i.e., 2 to 9). When using electronic microscopes toobserve and study muscle structure, researchers dis-covered two important characteristics of muscle fiber:(1) the muscle fiber depicts the horizontally bright anddark stripes in the regular arrangement (namely thestructure of horizontal stripes), and (2) muscle fiber isactually formed by finer fiber units that are perpendic-ular to the main fiber. Using electronic microscopes tostudy atomic structure of nucleic acid, observers canidentify the linear status inside the nucleic acid atomstructure with diameter around 20A◦. These examplesindicate that the application of electronic microscopes


significantly expedites the possibility of exploring thesecret of life and also plays an important role in theadvance of medicine, as well as the improvement ofhuman health [27].

In pathology, electronic microscopes have showedtheir importance in many applications. For example,electronic microscopes have been applied to patho-logical diagnosis of renal biopsy specimens. Renalglomerular disorder is a commonly diagnosed diseasein clinical practice and can be classified as primaryand secondary types of disorders. In order to moreaccurately diagnose the pathology of renal glomeru-lar disorder, renal needle biopsy often is used. Besidesdistinguishing the different patterns of the morpholog-ical and tissue elements inside glomeruli, electronicmicroscopes can be used to observe and detect micro-structural aberration or changes, especially in epithelialcells, mesenterium, base membrane, and interstitialsubstance, which cannot be observed or detected usingthe conventional optical microscopes. By analyzing theobserved micro-changes, physicians can find whetherthere are electron-dense deposits and correspondinglocations. However, the single electronic microscopictechnology also has limitation. In order to makerenal needle biopsy more useful and accurate in thepathology diagnosis, the combination of multiple tech-nologies, including the optimal use of conventionaloptical microscopes, immune-fluorescence micro-scopes, and electronic microscopes, is needed [23].

Using the electronic microscope not only hasimproved and enhanced the knowledge of the pathol-ogy, but also has provided a reliable foundation for thediagnosis of many diseases. With the rapid advanceof science and technology, electronic microscopeshave been and will continue be extensively usedin pathologic diagnosis. However, single electronicmicroscopic technology has some limitations in patho-logic diagnosis. For example, the field of vision (FOV)is reduced substantially due to the high spatial resolu-tion of the electron microscope. Each FOV typicallycan only show a single cell or the partial structure ofthe cell. Meanwhile, observers can only observe stilland dead specimens instead of the cells in vivo. There-fore, while electronic microscopes have been usedactively in clinical practice, one also needs to combineother technologies such as the quantitative analysisof the morphology of the micro-structural changesusing image processing, the nucleic acid hybridiza-tion in situ technology, and the apoptotic indices ofthe observation using electronic microscopes. Mean-

while, the scanning tunnel microscope and the atomicforce microscope also have been introduced recentlyinto the pathological area to improve the electronicmicroscopic technology. With closer connections tocomputer technology, the observers (i.e., pathologists)more easily and conveniently can use and control elec-tronic microscopes through the connected computerand networking systems.

4.3. Fluorescence microscopes

Irradiated by the short wavelengths of light (e.g.,ultraviolet and violet blue light 250 nm–400 nm), somesubstances can be excited by absorbing the energy andemit a longer wavelengths of light with energy degra-dation, which includes blue, green, yellow, or red lightwithin the wavelength between 400 nm and 800 nm.This is called photoluminescence [28]. Some spec-imens naturally can generate fluorescent light whenthey are illuminated by the light with the proper wave-length. For example, most lipid and protein can emitfluorescent light in blue. This phenomenon is calledauto fluorescence or primary fluorescence. However,most substances generate fluorescence light only whenthey are dyed with fluorochromes and are exposed inthe light of short wavelengths.

In the 1930s, florescent staining was applied toinvestigating and observing the morphology of bacte-ria, mildew, cells, and fiber. For example, Mycobacte-rium tuberculosis can be detected in sputum byacid– fast bacteria staining methods. In the 1940s, thefluorescent staining protein technology was inventedand applied extensively to the routine clinical immuno-fluorescence staining, which can examine and locatevirus, bacteria, mildew, protozoan, parasite, antigen,and antibody in animals and humans. It has been usedto investigate the pathogenesis and the cause of dis-eases such as categorizing and diagnosing glomerulardiseases and identifying the relation between humanpapillomavirus (HPV) and the cervical cancer. Thefluorescence microscope, a special tool for detectingfluorescence, has been getting more and more appli-cations in the clinical research and disease diagnosis[29]. The coupling system between a fluorescencemicroscope and a fluorescence spectrometer has beenextensively used in cell biology, biochemistry, phys-iology, neurobiology, and pathology. These systemscan provide qualitative and quantitative analysis forvarious structures in either living and fixed cells or tis-sues. Fluorescence microscopes can observe cells and


structures either emitting auto fluorescent or stainedby the fluorochromes. The light source of a fluorescentmicroscope is a high-voltage mercury lamp that emitsthe ultraviolet light with short wavelengths. The flu-orescent microscope also is installed with excitationand dichroic filters. The fluorochromes in the spec-imens are excited by the ultraviolet light and emitlight with different colors. This enables one to analyzethe distribution and locations of fluorescence insidethe cells or structures. Some tissues can emit autoor spontaneous fluorescent light. For example, lipo-foscin inside nerve or myocardial cells emit brownfluorescent light; Vitamin A of epithelia cells in hepaticIto and retinal pigment emit green fluorescent light;and monoamine (e.g., Catecholamine, 5 – HT, His-tamine) inside neuroendocrine cells and nerve fibersemits light with different colors under formaldehyde.Quinine and Tetracycline inside the tissues also emitfluorescent light. Some tissues inside the cells can bindwith fluorescein to emit fluorescent light. For example,Ethidium bromide and Acridine orange can combinewith DNA to measure the content of DNA. The flu-orescence microscope also has been widely used inthe immunocytochemistry research. By using fluo-rochrome to mark Isothiocyanate and Rhodamine, themarked antibody directly or indirectly can bind withthe corresponding antigen. Thus, clinicians can detectthe existence and distribution of antigen.

Since the invention of first fluorescence microscopein 1908, fluorescence detection has been improvingconstantly. This includes the introduction of high-voltage mercury lamps and the development of themulti-layer coating technology that is able to manu-facture the high quality excitation and cut-off filters.With high quality optical components, the fluorescentmicroscope made a great contribution to the devel-opment of immune-fluorescence in the 1980s. Inthe 1990s, the laser scanning confocal fluorescencemicroscopy was developed. It measures and deter-mines the fluorescence distribution within cells andtissue biopsies and obtains real-time overlappingphase-contrast and fluorescence images, as well as theprimary color images labeled with double fluorescents.Currently, more advanced technologies related to thesingle-photon fluorescence microscopy, two-photonfluorescence microscopy, 4 pi confocal fluorescencemicroscopy [30], and full internal reflection fluo-rescence microscopy [31] (total internal reflectancefluorescence microscopy) have been developed. Asa result, using these new tools, the technology and

application of fluorescence detection can be applied orimplemented in more research and clinical applicationsin pathology and other biomedical fields.

5. Digital microscope imaging

Although microscopy plays a very important role inbiology and medicine, the observation is performedwith the naked eye under the conventional opticalmicroscopes. This can result in eye fatigue after a rela-tively long time of continuous observation. In addition,the image information cannot be stored and processedfor different image enhancement purposes. To solvethese limitations, digital microscopes have been devel-oped and tested. A digital microscope (or imagingsystem) is an integrated design that combines tra-ditional optical microscope, digital multimedia, anddigital processing technology [32, 33]. As shown inFig. 6, a digital microscope imaging system typi-cally includes three components: Microscopy opticalmodule, data acquisition module, digital image pro-cessing, and software control modules. Among thesemodules, the optical module realizes the functionof the microscopic imaging; data acquisition modulerecords the images produced by digital video devices,including CMOS, CCD, digital camera stored in opti-cal module in the digital format, and then transfersthese digital images to the computer storage devicesthrough different graphics card interface or USB inter-face; and the software control module, the core ofthe whole system, controls the image capture, pro-cessing, and measurement in real-time to optimallyimprove the image quality. The digital images canbe monitored in real-time using a color TV or com-puter monitors. After using a variety of digital imageprocessing methods, digital microscopic imaging sys-tems more sensitively can capture and display theimage details. In fact, installation of online imageacquisition, processing, and analysis systems havebecome an important symbol of modern advancedmicroscopes.

Due to its significantly technological advantages,microscopic digital imaging technology also has beenused and/or integrated in a variety of electronic micro-scopes. For example, unlike the traditional electronmicroscopy imaging that uses electronic sensitive floorto generate photographic images, the digital micro-scope equipped with the CCD imaging system convertselectrons that carry image information through electro-


Industrial Cameraor Digital Camera

Power PlatformControl

OpticalMicroscope

Image Analysis System

Analysis ReportOutput

Fig. 6. The configuration of modern digital microscope imaging system.

1

2345

6

78 9

10

11

12

13

14

Fig. 7. The working principle of digital microscopic imaging system. 1 – Electron microscope lens; 2 – Fluorescence plate; 3 – Prism; 4 – PrismBlock; 5 – Drive screw; 6 – Motor; 7 – Base; 8 – Aperture; 9 – Prism; 10 – Lens; 11 – Lens; 12 – CCD; 13 – Image acquisition card; and 14 –Computer.

optical conversion devices to the light signal and sendsthe signal to the CCD. After the images are capturedwith CCD, they can be sent to the computer through animage acquisition card to perform a variety of requiredimage processing procedures (as shown in Fig. 7).

Thus, digital microscopic imaging technology is anextension of conventional microscopy. It integrates themicroscopy, image acquisition, and computer controland processing technology to manage and control thewhole imaging process, including image acquisition,sampling, processing, and data storage. Among these,the image acquisition and processing technology is thecore for digital microscopic imaging technology.

Current digital image acquisition devices can bedivided into three categories, including the use of (1)

analog cameras plus video capture card, (2) con-sumer grade digital cameras, and (3) professional gradedigital cameras. These three devices have their owncharacteristics and application fields in which pro-fessional grade digital cameras are the most popularchoice in digital imaging microscopy systems. Theintroduction of digital imaging technology creates agreat opportunity for the post-processing of acquiredimages. In particular, with the rapid advance ofmore powerful computers, digital microscopic imagescan be more effectively and efficiently processed andanalyzed.

Image analysis and processing may be custom-designed or may use sophisticated commercial micro-scopic image processing software packages. Many


professional software packages are well designedwith variety of useful functions or schemes. Theycan provide both simple geometric measurement andsophisticated analysis to identify the relationshipbetween the complex geometric structures. By provid-ing the absolute space calibration to ensure the mostaccurate measurements and the advanced image seg-menting technology, these commercial schemes canhelp observers to better distinguish the overlappingobjects, detect the contours and shapes of the smallobjects, identify similar objects or groups, and catego-rize or mark the different objects with different colors.Some schemes can be used as advanced analyticaltools that use the spectrum diagram, spectral profile,pseudo-color, three-dimensional surface shape, andother methods to manipulate the data and display it indifferent formats.

The advantage of connecting the microscope to acomputer is that it produces digital images. Using thesedigital images, the research scientists and clinicianscan apply various image processing and analysis soft-ware to obtain needed experimental data for variety ofpurposes. For example, using digital microscopes, onecan replace the manual process of counting red tideorganisms, a very tedious and time-consuming task.The computerized program is able to count the totalnumber of red tide organisms depicted in a sample andclassify red tide organisms in different size groups. Themagnified red tide organisms can be displayed on thecomputer screen. As a result, by combining with man-ual identification, this computerized method greatlyreduces the labor intensity of manual count using aconventional optical microscope.

6. New development in microscopic imagingtechnology

With the advance of technology and the specialrequirements of research projects, many new micro-scopic imaging methods have been developed andtested that aim to acquire the images with highresolution and large field of depth, as well as in three-dimensional stereoscopic format. As a result, usingthese new microscopic imaging systems and acquiredimages, the clinicians are able to conduct dynamic,non-destructive, and non-intervention in vivo (realtime) tests in the biomedical research and clinicalpractice. Following are several new developments inthis field.

6.1. Coexistence of a large field depth and highmagnification power

In the conventional optical microscope, a large depthof field cannot coexist with high magnification power.As a result, due to the limited depth of field, the opticalmicroscope cannot capture clear images of the targetedsmall objects located at different depths of the samplesurface. The use of a scanning electron microscopy(SEM) or a confocal microscopy can resolve the con-tradiction between magnification and depth of field.As a result, one can observe clear (sharp) images withlarger depth of field.

The SEM uses a very thin electronic beam to scanthe sample surface. Electrons produced by scanning arecollected by a specially designed detector. The corre-sponding electrical signals are further delivered to theCRT monitor screen to generate the three-dimensionalimage of the sample surface. These images also canbe captured into the pictures. The resolution of theSEM lies between optical microscopy and transmis-sion electron microscopy (TEM) (i.e., up to 3 nm).Meanwhile, the depth of field of the SEM is a hun-dred times higher than the optical microscope and atleast ten times greater than the TEM. Thus, it is ableto acquire a clear picture with a large depth of field.

The confocal microscope is an integrated tech-nology that combines the optical and digital imageanalysis technique. It is a new microscopic imagingtechnology specifically useful in acquiring the imagesof non-planar samples. It can obtain enormous depthof field and exquisite details and also can acquiretrue-color information that often cannot be obtainedusing an electron microscope. When using the confo-cal microscope, one first adjusts the focus to searchfor and reach the samples distributed in different depthlevels, captures all the images distributed in these lev-els with digital imaging devices, and transfers themto a computer (each of these aspects of the imagehas a clear location different from other images thatrecord different parts of sample morphology and colorinformation). After data analysis and integration usingthe special image processing software, the final highquality and clear picture is produced.

6.2. Three-dimensional imaging

The modern microscopy is not only limited toobserving the specimen surface. By combining laserand confocal microscopy, researchers and clinicians


can acquire and view the images of the cross sectionsof specimens at different depth levels. Then, usingcomputerized image processing and 3D reconstruc-tion algorithms, the three-dimensional shape profile ofspecimens can be acquired in high-resolution.

The main principle of laser scanning confocal micro-scope (LSCM) is to let the laser beam pass througha pinhole to generate a point light source. The laserlight passes through the excitation filter and arrivesat the beam splitter. Because the beam splitter canreflect excitation light with a shorter wavelength andtransmit the light with a longer wavelength, the excita-tion light is reflected at the beam splitter and transmitsthrough the objective lens. The laser beam scans dif-ferent focal planes marked by the fluorescent agentsunder the control of the scanning control device. Theexcited emission light from the fluorescent markspasses through the original incident light path anddirectly returns to the beam splitter. After passingthrough the transmission filter for the specific wave-length, the emission light finally reaches the detectionpinhole of the photomultiplier tube (PMT). By convert-ing the PMT current into the digital signals, an image iscreated and displayed on the computer monitor screen.

Since LSCM can continuously scan the living cellsand tissue or cell biopsy samples layer by layer toobtain images at different depth levels, it is alsonamed as “non-destructive optical biopsy”. The dis-tance between two LSCM scanned layers can be lessthan 0.1 um. Using computerized reconstruction algo-rithms, one can generate or acquire three-dimensionalimages that are able to observe or access the sophisti-cated cytoskeleton, chromosomes, organelles, and cellmembrane.

6.3. In vivo detection

Observing and monitoring living cells can be veryuseful in many research applications, but it is a dif-ficult technical challenge in the microscopic imagingfield. To enable researchers or clinicians to observe thedetailed structures of the specimens and also dynami-cally monitor the function of living cells or tissues inreal-time using microscopic pathology images, severalnew technologies have been developed and tested. Forexample, by applying the specific fluorescence probesto label the material to be observed, the researcherscan use LSCM to monitor dynamically the whole pro-cess of change in different locations or tissues of thecells after receiving stimulation in real time. LSCM

also can be used in many applications such as assayingthe changes within the PH of the cells, detect poten-tial changes in membranes, detecting the productionof intracellular reactive oxygen, testing the process ofdrugs into the tissue or cell membrane and locating itsposition, measuring the fluorescence resonance energytransfer (FRET), and real-time monitoring many otherliving cells.

7. Summary

Pathological imaging technology is a fast growingarea of academic investigations, industrial develop-ments and clinical applications. This paper presents abrief review of the fundamental concepts and a discus-sion on the recent developments and clinical potentialof contemporary microscopic imaging techniques. Theconventional optical microscopy has been an impor-tant tool in clinical pathology. The interface of opticalmicroscopy with digital image acquisition methodscombines the power of optical imaging, electronicdetection, and computerized analysis. These and otheremerging technologies will continue to evolve, enablecellular-, molecular-, and genetic-imaging, as well astissue imaging with high efficiency and accuracy, tofacilitate clinical screening and diagnosis.

Acknowledgements

The authors would like to acknowledge the sup-port in part of the Charles and Jean Smith ChairEndowment fund, and the support in part of NIH grantRO1CA136700.

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