Bancroft's Theory and Practice of Histological Techniques E-Book microscopy.pdf

Light microscopy John D. Bancroft 3 Introduction...

Light microscopy John D. Bancroft 3 Introduction The lens system of the light microscope allows the eye to see an image of the target tissue at varying This is an introduction to the theory of light micros- magnifications depending on the objectives used. copy. The subject is dealt with in more depth in The varying lenses seen in the modern microscope the previous editions of this book and further are present within the substage condenser below the information may be found in dedicated texts to slide, as well as above it. The additional objective the subject. lenses above the sample can be brought into posi- The light microscope is an essential part of the tion depending on the tissue magnification required. histopathology laboratory as it is the device with The objectives are usually mounted in a rotating disc which histological preparations are studied. The and are brought into alignment with the main body designs and specifications of modern microscopes tube of the microscope to select higher or lower vary widely, but the basic principle is the same as magnifications. the original simple microscope which used sun- The different magnifications required are achieved light as its light source (Fig. 3.1). Electric bulbs by altering three variables; firstly, the angle at which or light emitting diodes (LEDs) are now used to the light strikes the lens, the angle of incidence; produce a beam of light which is focused on the secondly, the curvature of the lens and finally, the tissue section or sample, and then the transmitted density of the glass or refractive index (RI). Parallel light passes through a set of objectives, along the light entering a lens from a small object is brought tube and through the eyepiece into the eye of the to a sharp focus at a point behind the lens, then the microscopist. eyepiece allows a magnified real image to be formed below the eyepiece (Fig. 3.2). This is the basic prin- ciple of light microscopy. Light and its properties Visible light occupies a narrow portion of the elec- tromagnetic spectrum and can be detected by the human eye, although the full spectrum extends from radio and microwaves through to gamma rays. Electromagnetic energy is complex, having both wave and particle-like properties. It is common practice to illustrate the light in the electromagnetic spectrum as a sine wave. The distance from one wave peak to another is the wavelength of Fig. 3.1 A standard modern light microscope. light (Fig. 3.3). 25  26 3 Light microscopy Fig. 3.4 The amplitude, i.e. brightness diminishes as light gets Eyepiece further from the source due to absorption into the medium through which it travels. The human eye responds to a complex mixture Real image of light of different wavelengths and when this approximates to the mixture of light derived from the sun, it is known as ‘white’ light. By definition, white light is a mixture of light which contains a percentage of wavelengths from all of the vis- Objective ible portions of the electromagnetic spectrum. One Object Virtual measure of the mixture of light given off by a light image source is color temperature. The higher the color tem- Condenser perature, the closer the light is to natural daylight derived from the sun. Light Light sources produce light in all directions and source usually consist of a complex mixture of wavelengths which define the color temperature of the light Mirror source. Some sources, e.g. tungsten filament and xenon lamps provide a relatively uniform mixture of wavelengths, although of different amplitudes Fig. 3.2 The light ray path through the microscope. The eye or intensities. Others, e.g. mercury lamps, provide sees the magnified virtual image of the real image, produced by the objective. discrete wavelengths scattered over a broad range, but with distinct gaps of no emissions between these peaks. Wavelength Most light sources are non-coherent, but standard optical diagrams draw light rays as straight lines Amplitude even though the actual light is emitted from the source in all directions. Another property important in understanding microscope optics is that some of the light is absorbed by the media (lens and air) through which it passes (Fig. 3.4). This produces a Fig. 3.3 Representation of a light ray showing its wavelength reduction in the amplitude, or energy level, of the and amplitude. light. The media can also have an effect on the actual speed of the light passing through the microscope, Light with a single wavelength is monochro- this is known as retardation. matic, but the majority of light sources are com- posed of many different colors and wavelengths Retardation and refraction which are refracted in different directions. The pan- Media through which light is able to pass will slow spectral distortion which can occur to an image can down or retard the speed of the light in proportion to be corrected by different types of lenses within a the density of the medium. The higher the density the microscope. greater the degree of retardation. Rays of light entering  Light and its properties 27 A B Denser C medium i a b r a Normal c Fig. 3.5 (a) Light rays passing from one medium to another at right angles to the interface are slowed down equally, i.e. retarded. (b) Rays passing at any other angle to the interface Focal point are slowed down in the order that they cross the interface and are deviated, i.e. refracted. (c) Rays passing through a curved lens are retarded and refracted. a sheet of glass at right angles are retarded in speed b but their direction is unchanged (Fig. 3.5a). When light enters the glass at any other angle, a change Fig. 3.6 (a) Light ray A shows the angles of incidence (i) and of direction also occurs and this is called refraction refraction (r). Ray B, entering the lens at an increased angle of incidence, is lost through the edge of the lens. Increasing the (Fig. 3.5b). A curved lens will exhibit both retardation angle of incidence further, Ray C shows total internal reflec- and refraction (Fig. 3.5c) and this is governed by: tion. (b) Parallel rays entering a curved lens are brought to a The angle at which the light strikes the lens, the common focus, the focal point. angle of incidence. The density of the glass, its refractive index. The curvature of the lens. normal, and when passing into a less dense medium it is refracted away from the normal. The angle of The angle by which the rays are deviated within incidence may increase to the point where the light the glass or other transparent medium is the angle emerges parallel to the surface of the lens – beyond of refraction and the ratio of the sine values of the this angle of incidence total internal reflection will angles of incidence (i) and refraction (r) gives a fig- occur, and no light will pass through (Fig. 3.6a). ure known as the refractive index (RI) of the medium (Fig. 3.6a). The greater the RI, the higher the density Image formation of the medium. The RI of transparent substances is Parallel rays of light entering a simple lens are important in the computation and design of lenses, brought together by refraction to a single point, the microscope slides, coverslips and mounting media. principal focus or focal point, where a clear image Air has a refractive index of 1.00, water 1.30 and will be formed of an object (Fig. 3.6b). The distance glass has a range of values depending on the type, between the optical center of the lens and the princi- mostly averaging 1.50. pal focus is the focal length. A lens has an additional Usually, light passing from one medium into pair of points, one either side of the lens, called con- another of higher density is refracted towards the jugate foci, and an object placed at one will form a  28 3 Light microscopy Screen White Object light Real image a Blue Red Fig. 3.7 A real image is formed by rays passing through the lens from the object, and can be focused on a screen. b Fig. 3.9 (a) Chromatic aberration. (b) Spherical aberration. Object Virtual brought to a shorter focus than red. This lens defect image is chromatic aberration (Fig. 3.9a) and results in an unsharp image with colored fringes. It is possible to construct compound lenses of different glass ele- Fig. 3.8 A virtual image is viewed through the lens but appears ments to correct this fault. An achromat corrected to be on the object side of the lens. for blue and red produces a secondary spectrum of yellow and green; this in turn can be corrected by clear image on a screen placed at the other. The con- adding more lens components, the more expensive jugate foci vary in position – as the object is moved apochromat. nearer the lens the image will be formed further Microscope objectives of both achromatic and away, at a greater magnification, and inverted. This apochromatic types are usually overcorrected for is the real image and is formed by the objective lens of longitudinal chromatic aberration and must be the microscope (Fig. 3.7). combined with matched compensating eyepieces If the object is placed nearer the lens, within the to form a good quality image. This restriction principal focus, the image is formed on the same on changing lens combinations is overcome by side as the object and is enlarged, the correct way up, using chromatic, aberration free (CF) optics which and cannot be projected onto a screen. This is the vir- correct for both longitudinal and lateral chro- tual image (Fig. 3.8) and is formed by the eyepiece of matic aberrations, and remove all color fringes. the microscope from the real image projected by the This is useful for fluorescence and interference objective. This appears to be at a distance of approxi- microscopes. mately 250mm from the eye, near the object stage Other distortions in the image may be due to level. This may be illustrated as in Fig. 3.2 with the coma, astigmatism, curvature of field and spheri- formation of both images in the upright compound cal aberration; they are due to the lens shape and microscope. quality. Spherical aberration is caused when light rays entering a curved lens at its periphery are refracted Image quality more than those rays entering the center of the lens and are not brought to a common focus (Fig. 3.9b). White light is composed of all the visible spec- These faults are corrected by making lenses of differ- tral colors and on passing through a simple lens, ent glass components, e.g. fluorite, and of differing each wavelength is refracted differently – blue is shapes.  The components of a microscope 29 The components of a microscope Object The light source A progression of light sources has been used from sunlight, oil lamps, low-voltage electric lamps and Condenser now LEDs. The latter operate via a transformer and can be adjusted to the intensity required. Condensers Light from the source is directed into the first major optical component, the substage condenser either directly, or via a mirror or prism. The con- Light source denser focuses or concentrates the available light Fig. 3.10 The function of the condenser is to concentrate, or into the plane of the object (Fig. 3.10). Generally, focus, the light rays at the plane of the object. the more light at the specimen, the better the image resolution. B B Condensers in microscopes are capable of verti- cal adjustment to allow for the varying heights or Objective thickness of the slides and once the correct position A A has been established it should not be moved, as any alteration will change the light intensity and impair the resolution. Condensers are usually provided with adjustment screws. The screws allow centering Object of the light path which should be routinely checked stage before using the instrument. The diameter of the light beam can be controlled via the aperture dia- phragm of the condenser. Adjustment of the iris diaphragm will alter the size and volume of the cone of light focused on the Condenser object. If the diaphragm is closed too much, there is Aperture increased contrast and the image becomes refrac- diaphragm tile. Leaving the diaphragm wide open will cause the image to suffer from glare due to extraneous light interference. In both cases the resolution of the A B B A image is poor. The correct setting for the diaphragm is when the numerical aperture of the condenser Fig. 3.11 Light rays A illustrate the ‘glare’ position resulting in is matched to the numerical aperture of the objec- extraneous light and poor resolution whereas B indicate the correct setting of the substage iris diaphragm. tive in use (Fig. 3.11) and the necessary adjustment should be made when changing from one objective to another. This is achieved by removing the eye- The iris diaphragm should not be closed to piece, viewing the substage iris diaphragm in the reduce the intensity of the light, either use filters or back focal plane of the objective, and closing it down alter the rheostat setting of the lamp transformer. to two-thirds of the field of view. With experience In condensers fitted with a swing-out top lens, the correct setting can be estimated from the image this is turned into the light path when the higher quality. power objectives are in use. This focuses the light  30 3 Light microscopy into a field more suited to the smaller diameter of possible from the object, forming a high-quality the objective front lens. Swing the top lens out of magnified real image above the lens unit. the path with the lower power objectives, or the The magnification power is usually inscribed on field of view will only be illuminated at the center. the side of the objective lens and is a reflection of Aplanatic or a highly corrected achromatic substage the object: image ratio – most microscopes have 1:4, condenser should be used with an apochromatic or 1:10 and 1:40 as the basic minimum. The ability of an fluorite objective. objective to resolve detail is indicated by its numeri- cal aperture (NA), and not its magnifying power. This Object stage is expressed as a value calculated by a mathemati- This sits above the condenser and supports the glass cal formula. The NA is the product of the refractive slide. It is perpendicular to the optical path with an index of the medium between the lens and objective, aperture for the light. The stage moves in two direc- and the sine of the angle between the optical axis of tions and Vernier scales enable the operator to return the lens and the outermost ray of light which can to an exact location on the specimen. The scale is also enter the front of the lens. The maximum NA attain- used for measuring the separation of various ele- able for a dry objective is 0.95, for water it is 1.3 and ments in the plane of section. oil 1.5. Resolution is the smallest distance between two Objectives dots or lines which can be seen as separate entities The type and quality of the objective has the great- and is dependent on the wavelength of the light est influence on the performance of the microscope. used and the NA of the lens. The resolving power There may be from 5 to 15 lens elements within the of the objective is its ability to resolve the detail objective depending on the image ratio, type and which can be measured, i.e. as the NA of an objec- quality (Fig. 3.12). The main task of the objective is tive increases, the resolving power increases but to collect and unite the maximum amount of light working distance, flatness of field and focal length decrease. Objectives are available in varying quality and types (Fig. 3.12.). Achromatic objectives are the most widely used for routine purposes; the more highly corrected apochromats, often incorporating fluorite glass, are used for more critical work, and plan-apo- chromats, which have a field of view which is almost perfectly flat, are recommended for photomicrogra- phy and cytology screening. Objectives are designed for use with a coverglass protecting the object and a value giving the correct coverglass thickness, usually 0.17mm, is engraved on it. Apochromats between 40:1 and 63: 1 require the coverslip thickness to be precise and some are mounted in a correction mount which can be adjusted to suit the thickness of the coverglass used. The body tube and eyepiece Achromatic Apochromatic The image from the objective is formed in the body Fig. 3.12 Diagram of achromatic and apochromatic objectives. tube and magnified by the eyepiece, presenting the  Phase contrast microscopy 31 eye with a virtual image which appears to be in the same plane as the object, but this produces uneven plane of the object. This image is observed at an opti- illumination. Köhler illumination is used for photog- cal distance 250mm from the eye. The body tube can raphy and more specialized microscopes where an be monocular, binocular or combined with photo- image of the light source is focused by the lamp graphic imaging tubes. collector or field lens in the focal plane of the stage condenser on the aperture diaphragm. The image Using the microscope of the field or lamp diaphragm is focused in the object plane and the illumination is even (Fig. 3.13). The microscope should remain clean and well-­ The illumination must be centered with respect to maintained. Dust, finger prints and other materials, the optical axis of the microscope to prevent poor e.g. fragments of glass, whether on the substage, resolution. eyepiece or the gearing mechanism will impede the performance of any microscope. Only appropri- Dark field illumination ate oils should be used on the stage to allow free Occasionally it is preferable, or essential, that movement of the object in two dimensions. The unstained sections or living cells are examined. substage condenser and objectives should be freely These specimens and their components have mobile, but not loose. Only appropriate cleaning refractive indices close to that of the medium in cloths for the lenses should be used, cleaning any which they are suspended and are difficult to see oil immersion lens after each use. The light source by bright field techniques because of their lack of should be appropriate for the microscope and cen- contrast. Dark field microscopy overcomes this by tered if necessary by adjusting the condenser posi- preventing direct light from entering the front of tion. The filters used will depend on the type of the objective and the only light gathered is that microscopy. Coarse and fine focusing are achieved reflected, or diffracted by structures within the by moving the top tube and condenser lenses specimen (Fig. 3.14). This causes the specimen to towards or away from the section on the slide appear as a bright image on a dark background, being careful not to crush the object lens into the the contrast being reversed and increased. Dark slide itself! When not in use the microscope should field microscopy permits the detection of particles be covered. smaller than the optical resolution obtained in bright field microscopy due to the high contrast Magnification of the scattered light. Many small structures are more easily visualized by dark field techniques, In a standard microscope with an optical tube length although the resolution may be inferior to bright of 160mm, total magnification is the product of the field microscopy. It is particularly useful for spi- magnification values of the objective and eyepiece. rochetes, flagellates, cell suspensions, flow cell Using the combined magnifications of the objective techniques, parasites and autoradiographic grain (40x), tube length (1.25x), and eyepiece (10x), it is counting. Thin slides and coverglasses should be possible for a total magnification of 500x being seen used and the preparation must be free of hairs, dirt, by the observer. and bubbles. Illumination Phase contrast microscopy There have been varying approaches to maximize Unstained and living biological specimens have little the illumination within the microscope. Critical illu- contrast with their surrounding medium, although mination is used in simple equipment where the light small differences of refractive index (RI) do exist source is focused by the substage condenser in the in their structures. Phase contrast overcomes these  32 3 Light microscopy Diaphragm Object plane Source Condenser Objective Eyepiece a Aperture Object Back focal diaphragm plane plane Collector Source Condenser Objective Eyepiece Field b diaphragm Fig. 3.13 (a) Critical illumination. (b) Köhler illumination. Objective problems by using controlled illumination with the Direct rays full aperture of the condenser and therefore improv- ing resolution. The higher the RI of a structure, the darker it will appear against a light background, i.e. with more contrast. To achieve phase contrast, a microscope requires Specimen modification of the objectives and condenser, the specimen to retard light by between 1⁄8 and 1⁄4 of the light wavelength (λ) and an intense Köhler illumina- Condenser tion light. Usually the microscope condenser carries a series of annular diaphragms made of opaque glass with a clear narrow ring which produce a controlled hol- low cone of light. Each objective requires a different size of annulus, and an image of this is formed by the condenser in the back focal plane (BFP) of the objective as a bright ring of light. The objective is modified by a phase plate which is placed at its BFP Fig. 3.14 In dark-field illumination no direct rays enter the (Fig. 3.15). A positive phase plate consists of a clear objective: only scattered rays from the edges of structures glass disc with a circular trough etched in it to half within the specimen form the image (dashed lines). the depth of the disc. The light passing through the  Polarized light microscopy 33 E annulus and objective phase plate will require cen- D tering. When the hollow cone of direct light from the annulus enters the specimen, some of the cone will pass through unaltered, whilst some rays will C be retarded or diffracted by approximately ¼ λ. The majority of the direct light will pass through the trough in the phase plate, whilst the diffracted rays pass through the thicker clear glass and are further retarded. The total retardation of the diffracted rays is now ½ λ and interference will occur when they are recombined with the direct light. A contrasting image is achieved, revealing small details within unstained cells. This is a quick and efficient way of examining unstained paraffin wax, resin and frozen sections, as well as studying living cells and B their behavior. Interference microscopy In phase contrast microscopy the specimen retards some of the light rays with respect to those passing through the surrounding medium. The resulting interference of these rays provides image contrast A but with an artifact called the phase halo. In the inter- ference microscope the retarded rays are entirely separated from the direct or reference rays, allow- ing improved image contrast and color graduation. Quantitative measuring of phase change (optical Fig. 3.15 The annulus, A, is at the focal plane of the condenser, B is the object plane and C the phase plate at the BFP. Light path difference), refractive index, dry mass of cells rays D are diffracted and retarded by the specimen with a (optical weighing), and section thickness are also total retardation of ½λ compared with the direct light, E, which improved. is unaffected by the specimen. Polarized light microscopy trough has a phase difference of ¼ λ compared to the rest of the plate. The trough also contains a neu- The use of polarized light in microscopy has many tral density, light absorbing material to reduce the useful and diagnostic applications and it is recom- brightness of the direct rays, which would otherwise mended that cellular pathology laboratories have a obscure the contrast obtained. simple system of polarizing microscopy as a mini- It is essential that the image of the bright annu- mum. Numerous crystals, natural and artificial lar ring from the condenser is centered and super- fibrous structures, pigments, lipids, proteins, bone imposed on the dull trough of the objective phase and amyloid deposits exhibit birefringence, i.e. the plate. This is achieved by using either a focusing ability to ­produce plane polarized light. In histologi- telescope in place of the eyepiece, or a Bertrand cal material, birefringence is produced by asymmet- lens (a small convergent lens), situated in the ric particles too small to be resolved even by the best body tube of the microscope. Each combination of lenses.  34 3 Light microscopy The polarizing microscope is a conventional between the rays, and if they are recombined, microscope in which a Nicol prism or polariz- interference occurs and various spectral colors ing disc is interposed in the light path below the are seen. condenser. This polarizer converts all the light Originally polarizers made from calcite and passing through the instrument into plane polar- known as Nicol prisms, after their inventor, were ized light, i.e. light which vibrates in one optical cemented together with Canada balsam in such plane only. A similar second prism, the analyzer, a way that the slow ray was reflected away from is placed within the barrel of the microscope the optical path and into the mount of the prism, above the objective lens. When the analyzer is leaving only the polarized fast ray to pass through rotated until its axis is perpendicular to that of (Fig. 3.17). the polarizer, no light can pass through the ocu- The optic axis of a birefringent crystal is the direc- lar lens resulting in a dark field effect. The field tion in which light may pass unaltered. Substances will remain black if an isotropic or singly refrac- through which light can pass in any direction and tive object is placed on the stage. A birefringent at the same velocity are isotropic, and are not able to object however will appear bright upon the dark produce polarized light. Some substances and crys- background. tals can produce plane polarized light by differen- Light entering a birefringent crystal, e.g. calcite, tial absorption and give rise to the phenomenon of is split into two light paths, each determined by a dichroism. different refractive index (RI) and each vibrating The two phenomena detected in polarized light, in only one direction, i.e. polarized but, at right birefringence and dichroism, are of value to the his- angles to each other (Fig. 3.16). The higher the tologist. A dedicated polarizing microscope uses two RI, the greater the retardation of the ray, so that polarizers (Fig. 3.18). One, always referred to as the each ray leaves the crystal at a different veloc- polarizer, is placed beneath the substage condenser ity. The high RI ray is called slow and the low RI and held in a rotatable mount, this can be removed ray is called fast. There is also a phase difference from the light path when not required. The other, the analyzer, is placed between the objective and the eyepiece. When a birefringent substance is rotated between two crossed polarizers, the image appears and dis- appears alternately at each 45° of rotation, i.e. in a Fig. 3.16 A birefringent crystal such as calcite can split a ray Fig. 3.17 A Nicol prism is constructed so that one part of the of light into two light paths, each vibrating at right angles to ray is allowed to pass whilst the other is directed away from the other. the optical path and is lost.  Polarized light microscopy 35 complete revolution of 360° the image appears and in intensity and color are seen during rotation is extinguished four times. When one of the planes to 90° and back to its original color in the next of vibration of the object is in a parallel plane to 90° (Fig. 3.19). This is due to the differential the polarizer only one part ray can develop, and absorption of light depending upon the vibra- its further passage is blocked by the analyzer in tion direction of the two rays in a birefringent the crossed position. However, at 45°, phase dif- substance. ferences between the two rays can develop and are Weak birefringence in biological specimens is able to combine in the analyzer to form a visible enhanced by the addition of dyes, e.g. Congo Red image. for amyloid, or impregnating metals in an orderly Some birefringent substances are also dichroic, linear alignment. Although only one polarizer is being capable of emitting two colors. Only the needed to detect the resulting dichroism, adding an polarizer is used and, if no rotating stage is avail- analyzer can enhance the image. able, the polarizer itself can be rotated. Changes 0° Eyepiece Blue Analyzer 90° Green Objective Stage 180° Blue Polarizer Lamp Mirror Fig. 3.19 When a dichroic substance is rotated in polarized light using the polarizer only, changes of color and intensity can be seen after rotating 90°. The original color returns after Fig. 3.18 A microscope equipped for polarized light. a further 90° rotation.  36 3 Light microscopy The sign of birefringence is diagnostically use- Transmitted light fluorescence ful and is determined by the use of a compensa- All light sources emit a wide range of wave- tor, i.e. a birefringent plate of known retardation, lengths, including the shorter ultraviolet and either above the specimen or below the polar- blue wavelengths of interest in fluorescence. izer at 45° to the direction of polarized light. If Only a few sources emit sufficient short wave the slow ray (higher RI) is parallel to the length light for practical use and originally the most of the crystal or fiber, the birefringence is posi- commonly used were high pressure mercury tive. If the slow ray is perpendicular to the long vapor or xenon gas lamps. Halogen filament axis of the structure, the birefringence is nega- lamps can produce enough light for some wave- tive. Rotating the compensator or the specimen length excitation in the blue and green range. The until the slow direction of the compensator is choice of a suitable source depends upon the type parallel to the long axis of the crystal or fiber of work to be performed. Traditionally, mercury turns the field red and if the crystal is blue the vapor burners were used for routine observation birefringence is positive. If the crystal is yellow, purposes, these operated on alternating current the slow direction of the compensator is parallel and their starting equipment was not expensive. to the fast direction of the crystal and the bire- However, they were toxic, required warming up fringence is negative. Quartz and collagen exhibit and the bulb needed to cool before further use. positive birefringence whilst Polaroid discs, These high pressure gas lamps have now been calcite, urates and chromosomes are negatively replaced by LED technology and the new LEDs birefringent. used in fluorescence microscopes do not have these problems. Fluorescence microscopy Preparations for fluorescence may contain other fluorescing material in addition to that in which one Fluorescence is the property of certain substances is interested. It is necessary to filter out all but the which when illuminated by light of a specific wave- specific excitation wavelength, to avoid confusion length will re-emit this light at a longer wavelength. between the important and the unimportant fluores- In fluorescence microscopy, the exciting radiation cence within the sample being examined. is usually in the ultraviolet wavelength, approxi- A variety of filters are available for this purpose. mately 360nm or, the blue region, approximately Dyed in the glass filters, e.g. UG 1 and BG 12 are 400nm, although longer wavelengths can be used broadband filters and transmit a wide range of with some modern dyes. wavelengths, the width of the range depending A substance which possesses a fluorophore will upon the composition and thickness of the filter. fluoresce naturally. This is known as primary fluo- It is better to employ filters of a narrower band rescence or autofluorescence. Ultraviolet excitation is transmission which have their transmission peaks required for optimum results with substances such closer to the excitation maximum of the fluoro- as vitamin A, porphyrins and chlorophyll. Dyes, chrome being used, e.g. FITC. Narrow band filters chemicals and certain antibiotics added to tissues are often of the interference type, and are vacuum- produce secondary fluorescence of structures and are coated layers of metals on a glass support. They called fluorochromes. The majority of fluorochromes have a mirror-like surface, and must be inserted require only blue light excitation and this is the most in the beam with their reflective face towards the common use of fluorescence in microscopy. Induced light source. fluorescence is a term applied to substances such as Barrier or suppression filters are placed before catecholamines which, after treatment with formal- the eyepiece to prevent short wavelength light dehyde vapor, are converted to fluorescent quino- damaging the retina of the observer (Fig. 3.20). line compounds. However, they must allow the fluorescing color to  Fluorescence microscopy 37 pass, otherwise a negative result may be obtained. has a number of advantages over using the trans- Barrier filters are colorless, through yellow to dark mitted route. In principle, the excitation beam, after orange and are of specific wavelength transmis- passing the selection filters, is diverted through the sion, e.g. a K.470 filter will block all wavelengths objective, on to the preparation where fluorescence below 470nm. is stimulated. This fluorescence travels back to the Bright field condensers are able to illuminate the observer by the normal route (Fig. 3.21a). Dichroic object using all the available energy, but they direct mirrors have been produced which divide and the rays beyond the object into the objective which divert the beam and are therefore able to transmit is a potential hazard to the eyes of the observer. light of some wavelengths and reflect other wave- They can also set up disturbing autofluorescence in lengths (Fig. 3.21b). Selecting the appropriate mir- the objective itself. A dark field condenser is there- ror, the desired wavelength is reflected to the object fore used in most systems. This does not allow and the remainder passes through to be lost. The direct light into the objective and it is more likely visible fluorescent light is collected by the objective to give a dark contrasting background to the fluo- in the normal way, passes to the eyepiece and any rescence. Only about 10% of the available energy excitation rays bouncing back from the slide and is used and this is dependent on the design of the coverglass are reflected back along their original condenser. path to the source and prevented from reaching the Simple achromatic objectives should be used observer. The objective in this system also acts as a with bright field illumination to prevent auto- condenser, so the illumination and objective numeri- fluorescence. However, with dark-ground illu- cal apertures are the same, optically correct, and in mination, the range of objectives is considerably their most efficient condition. Fluorescence is stimu- widened, and more elaborate lenses with higher lated on the observer’s side of the preparation and is apertures and better light gathering power are therefore more brilliant, not being masked by cover- possible. ing material or section thickness. Any type of objective, including sophisticated Incident light fluorescence phase contrast and interference contrast objec- Incident illumination, or lighting from above and tives, can be used for simultaneous transmit- through the objective down to the object (Fig. 3.21) ted illumination with normal tungsten lighting, Heat filter Object Source Wavelength selection filter Barrier filter Condenser Objective Eyepiece Red stop filter Fig. 3.20 Light path for transmitted fluorescence. Light of all wavelengths pass from the source through a heat-absorbing filter, into a second filter which removes red light, and then through a wavelength selec- tion filter which allows only the desired excitation wavelength(s) to pass. On passing through the speci- men, the objective collects both exciting and fluorescent wavelengths. The former is removed by a barrier filter to protect the eye of the observer.  38 3 Light microscopy Eyepiece Source Suppression (barrier filter) Filters Mirror Incident fluorescence illumination Objective a Object Wavelengths transmitted Wavelengths reflected b Fig. 3.21 (a) Diagram of an incident fluorescence microscope layout. (b) Effect of a dichroic mirror. allowing the demonstration of both fluorescence digital image capture, and these images are mono- and the morphology of the preparation. This is chromatic, i.e. black and white images. The highly useful when normal stains cannot be used for fear colored fluorescence images which appear in pub- of masking any fluorescent reactions. Brighter lications are the result of pseudo coloring compos- images are seen if dichroic mirrors are used as up ite images. to 90% of the excited energy can reach the prepa- ration and 90% of the resultant visible light can The confocal microscope be presented to the eye. Oil and water immer- Using conventional epifluorescence microscopes in sion objectives in low and high powers have been fluorescence microscopy, the fluorochrome present developed, they have higher numerical apertures in the field of view will be excited whether it is in, and can gather more light, avoiding much of the or out of focus. The out of focus fluorescence will lost stray light reflected from coverslips. The use reduce the contrast and resolution of the image. of low magnification eyepieces improves fluores- The confocal system uses a pinhole stop to observe cence techniques. the specimen, excluding the out of focus portion The filters and light sources used in fluores- of the image. The axial resolution in the confocal cence microscopy in modern systems rely on system is greatly improved to 0.35mm in reflection,  Fluorescence microscopy 39 with additional small but important gains in lat- can be recombined to create a 3D image of a cell eral resolution. This method therefore lends itself or structure even when using multiple labeling to optical sectioning. With modern computer tech- techniques. nology and software, a series of optical sections