Microscopes And Optical Microscopy Techniques

Microscopes and Optical Microscopy Techniques

By

Thomas G. Rand Department of Biology Saint Mary’s University Halifax, Nova Scotia Canada, B3H 3C3 [email protected]

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Table of contents Microscopy Overview 1) Microscope Types Light Microscopes Stereomicroscopes: Bright field compound microscopes: Phase-contrast Microscopes: Differential interference contrast (DIC) microscopes: Dark Field Microscopes: Polarizing Microscopes: Fluorescence Microscopes:

Electron Microscopes Transmission electron microscopes: Scanning electron Microscopes: Scanning transmission electron microscopes:

2) General Principles of Light Microscopy Parts of Microscope: Ocular lens or eyepiece: Body tube: Nosepiece: Objective lenses: Immersion Media: Stage: How to use a Vernier Scale: Condenser: Iris Diaphragm: Focusing knobs: Light source:

Light, Lenses, Aberrations and Lens Corrections: 1) Light: Interference: Reflected light: Refracted Light: Refractive Index: Diffracted Light: 2) Lenses: Lens overview: Biconvex (++ or converging) lenses: 2

Biconcave (-- or diverging) lenses: Composite lenses: 3) Aberrations: Spherical aberration: Chromatic aberration: Other Lens Aberrations: Astigmatism: Field curvature: Comatic aberrations:

Numerical Aperture and Depth of Field Numerical Aperture: Depth of Field:

Magnification and Resolution: Magnification: Resolution:

Micrometry: Procedure:

Path of Light through compound microscope: Critical (or Nelsonian) Illumination: Köhler method of microscope illumination: Procedure for setting up Köhler illumination in bright field microscopes lacking a field diaphragm: Setting Up Köhler Illumination in microscopes with field apertures: Bright Field Illumination Set-up: Phase Contrast Illumination set-up: Dark-Field Illumination Set-up:

Care and Maintenance: Filter and lens cleaning: Storage:

References: Appendices: Appendix 1. Preparation of wet mounted specimens Appendix 2. Quantitative Microscopy. Invited lecturer Appendix 3. Photomicroscopy. Invited lecturer

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Microscopy Overview: Microscopy is the discipline that uses microscopes to view objects. Microscopes are the standard instruments with which to magnify and examine cell and histological preparations. They have been used to study biological material for over 400 years. However, the earliest evidence of magnifying glass forming a magnified image dates back to the Book of Optics published by Ibn al-Haytham (Alhazen) in 1021. The book was translated into Latin, by Roger Bacon who subsequently described the properties of magnifying glass in 13th-century England, followed by the development of eyeglasses in 13th-century Italy.[2] Van Leeuwenhoek (1632-1723) has often been credited as being the first to use microscopy for the study of biological material and to record microscopic observations of muscle fibers, bacteria, spermatozoa and blood flow in capillaries. In basic design, probably all of Leeuwenhoek's instruments -- certainly all the ones that are known -- were magnifying glasses. Van Leeuwenhoek's microscopes consisted of a small, single converging lens mounted on a brass plate, with a screw mechanism to hold the sample or specimen to be examined.

Figure 1. Illustrations of one of Leeuwenhoek's "microscopes". Compared to modern microscopes, the microscope used by Leeuwenhoek is an extremely simple device, using only one lens, mounted in a tiny hole in the brass plate that makes up the body of the instrument. The specimen was mounted on the sharp point that sticks 4

up in front of the lens, and its position and focus could be adjusted by turning the two screws. The entire instrument was only 3-4 inches long, and had to be held up close to the eye; it required good lighting and great patience to use. The development of the compound light microscope was in 1590 by the Janssens, manufacturers of eyeglasses, some forty years before Leeuwenhoek was born. Their development opened the door to the microscope world. The Janssens microscope magnified objects up to 20 to 30 times their original size. Several of Leeuwenhoek's predecessors and contemporaries, notably Robert Hooke in England and Jan Swammerdam in the Netherlands, had also built compound microscopes and were making important discoveries with them. Perhaps Hooke’s most famous microscopical observation was from his study of thin slices of cork which was later articulated in cell theory that cells are the fundamental unit of all living organisms.

Figure 2. Hooke’s cork illustration In "Observation XVIII" of the Micrographia, he wrote: . . . I could exceedingly plainly perceive it to be all perforated and porous, much like a Honey-comb, but that the pores of it were not regular. . . . 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. . . The compound microscopes used by Hooke and his contemporaries were much more similar to the microscopes in use today than those employed by Leeuwenhoek.

Microscope Types There are two basic classes of microscopes used in laboratories today. These are the light and electron microscopes.

Light Microscopes: a) Stereomicroscopes: 5

Stereomicroscopes, also called dissecting microscopes, play useful roles in laboratories that examine biological material and can provide a useful link between unaided and higher power examinations. They can be extremely useful to histologists for the identification of tissue and its orientation before it is processed for conventional histology using compound light microscopy. Stereomicroscopes are essentially two modified compound microscopes which focus on the same point from slightly different angles (15° from perpendicular). This allows the specimen to be viewed in three dimensions. As opposed to conventional compound microscopes, the image is upright and laterally correct (not upside down and backwards) (see below). Stereomicroscopes are relatively low power compared with compound microscopes, and magnification is usually below 100x. They can have a single fixed magnification, several discrete magnifications, or a zoom magnification system. Working distance (see below) is much longer, usually between 10 to 15 cm, than with a typical compound microscope as well, allowing work to be done on the specimen while it is being observed through the microscope (hence the name "dissecting microscope"). Many stereomicroscopes are modular in design allowing a variety of stands, eyepieces, objectives, and lighting techniques to be implemented depending on the intended use. Stereomicroscopes can also employ either transmitted from below or reflected light from above the specimen. Transmitted light can provide some detail on the internal structure, while reflected light provides some surface detail of the specimen. b) Bright field compound microscopes:

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Bright field microscopes employ the simplest form of microscopy in which light unaltered by either polarizers or filters, is either transmitted through or reflected from the specimen. All light microscopes are capable of bright field imaging. Upright (those microscopes with illumination below the specimen) bright field microscopes are probably the most widely used for observing stained or naturally pigmented or highly contrasted tissue, smeared or filmed specimens associated with histology, pathology, anatomy, cytology and/or mycology, among others.

Figure a-b. a) Stained section of dense bone showing Haversian and Volkmann’s canals; b) Air sampled particles showing epithelial cells and a muriform fungal spore (Alternaria alternata).

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However, bright field microscopy is not routinely employed for observing unstained, low contrast specimens or living cells (unless they have been vitally stained), as light will pass through them without delineating structural detail. Observing these types of specimens is best achieved using either phase-contrast or differential interference contrast microscopy. c) Phase-contrast Microscopes: Light microscopes can be equipped with a Universal condenser (see below) that supports filters, which can accentuate differences in light as it passes through living specimens (see below). These types of light microscopes are said to be phase contrast microscopes. Phase-contrast microscopy exaggerates the small differences in refractive indices (RI) of living biological material to provide structural detail.

RI exaggeration is achieved by advancing or retarding light waves, thus converting them into differences of amplitude which are seen as variations in brightness. Two rays of light striking the same point of a screen will either reinforce or interfere with each other according to the relative positions of their wavelengths: light waves of similar phase will reinforce each other and double brightness; light rays that are retarded by exactly one half a wavelength will interfere or subtract from the other and produce no light (see below). Smaller phase differences will produce smaller alterations of amplitude and a picture will be built up of a pattern of different brightnesses.

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Figure a-b. a) Unstained amoeba viewed using bright field microscopy and b) phase contrast microscopy. Note improved structural detail associated with b). d)

Differential interference contrast (DIC) microscopes:

Differential interference contrast microscopy (DIC), also known as Nomarski Interference Contrast (NIC) or Nomarski microscopy, is an optical microscopy illumination technique used to enhance the contrast in unstained, transparent samples. DIC works on the principle of interferometry to gain information about the optical density of the sample, to see otherwise invisible features. A relatively complex lighting scheme produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo (see figure above). DIC works by separating a polarised light source into two beams which take slightly different paths through the sample. Where the length of each optical path (i.e. the product of refractive index and geometric path length) differs, the beams interfere when they are recombined. This gives the appearance of a three-dimensional physical relief corresponding to the variation of optical density of the sample, emphasizing lines and edges though not providing a topographically accurate image. DIC has strong advantages in uses involving live and unstained biological samples, such as a smear from a tissue culture or individual water borne single-celled organisms. Its resolution and clarity in conditions such as this are unrivaled among standard optical microscopy techniques.

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Figure .Components of the basic DIC microscope (after Wikipedia, the free encyclopedia).

Figure . DIC light path (after Wikipedia, the free encyclopedia).

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Figure. Differential interference contrast (DIC) microscopy revealing two isolated muscle fibers. N = nucleus; s = basic contractile unit of skeletal muscle is the sarcomere; single arrowheads = myofibrils; double arrowheads = endomysium with its occasional flattened connective tissue cells and reticular fibers.

Figure. Differential interference contrast (DIC) microscopy revealing Aspergillus condidiophore and Hulle cells.

e) Dark Field Microscopes: This form of microscopy is particularly useful for examining freshly prepared cells, especially bacteria and other micro-organisms, as well as unicellular parasites in body fluids. It can also be a valuable tool for evaluating thin, unstained whole mounted material and sections in fluorescent microscopy. In dark field microscopy, no direct rays of light from the condenser enter the microscope. Specimens are visible not because light passes directly through them but because they cause diffraction (bending of light around corners) and reflection of indirect light into the microscope (see below). Analogously, aerosolized dust is seen in sunlight due to diffraction of light from dust particle surfaces. The principle of dark field illumination is illustrated in the figure below.

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In this illustration a masking disk is placed below a parabolic condenser lens (see below) which prevents light transmission at the centre of the field of view. Light can only enter around the edge of the field. Examination of a clean slide will show the field of view to be black and empty. If the slide supports solid particles, these will cause light ray diffraction, refraction and reflection and some of these rays will enter into the microscope objective causing the particles to be brilliantly illuminated against a black background. A dark field condenser is constructed so that the alterations in light path takes place at a definite distance above it. This means that the slide and cover slip thickness are important determinants if clear specimen detail is to be obtained. If the slide and/or coverslip are too thick the field of view will be grey and contrast poor. Moreover, if the slide and/or coverslip are scratched, or marked with fingerprints, and if the mounting medium improperly dispensed with air bubbles, these will all show up. Clean preparations, free of dust, grease and scratches are essential for critical dark-field microscopy.

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Figures a-b. a) Dark field image of spirochaete bacteria; b) dark field image of bone formation in developing chick wing.

f) Polarizing Microscopes: The polarizing microscope takes advantage of 1) the electromagnetic properties of light vibrating in all directions at right angles to the path of the ray of light itself. In polarizing microscopy, the light waves are made to vibrate in one plane only. This is achieved by the use of two, interposing prisms of calcite (Nicol prisms) in the path of the beam of light. One prism is fitted below (polarizer) and the other above the specimen (analyzer). The analyzer prism must be able to rotate though 90°. When the diagonals of the prisms are parallel, polarized light passes through both. If the analyzer is rotated through 90°, the rays transmitted through the lower prism will not pass through the analyzer and the field is dark. Polarizing microscopy also takes advantage of 2): a) The isotropic (singly refractile) properties of objects. Isotropic objects do not change the direction of the beam and are not illuminated when examined using polarizing microscopy. These substances are most common. They include glass, certain crystals and most animal cells and tissues. b) The anisotropic (birefringent or doubly refractile) properties of objects. Anisotropic substances will alter the direction of light ray vibration when examined using polarizing light and will cause the substance to be bright against a dark background. Anisotropic substances include crystals such as talc, and vegetable fibres such as cotton and linen. Animal tissues that exhibit birefringence include collagen fibres in connective tissue, bone matrix, and striated muscle. Birefringence is also exhibited by some lipids such as cholesterol, and certain 13

pigments such as amyloid and those induced by fixation artifact (e.g. formalin pigment), amongst others.

Figures a-c. a) Amyloid deposits in connective tissue; b) bright field microscopy showing crystals in thyroid colloidal fluid; c) polarizing microscopy shows crystals to be birefringent calcium oxalate, which is one of the features of nodular goiter.

g) Fluorescence Microscopes: Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences.

Figure showing schematic of fluorescent microscope. (after Wikipedia, the free encyclopedia).

In epifluorescence microscopy, the excitatory light is passed from above, through the objective and then onto the specimen instead of passing it first through the specimen. A fluorescent specimen or fluorescently labeled regions of interest are illuminated using invisible ultraviolet light produced by high pressure mercury or xenon arc lamps; light rays of longer wavelength within the visible light spectrum are given off and these are 14

seen as various colors against a black background. Fluorescent techniques are widely used in animal and plant cell research. Because epifluorescent microscopy employs short wave ultraviolet light, resolution (i.e. ability to distinguish between two closely apposed points (see below)) is improved over other light microscopy techniques. In conjunction with immunological techniques, fluorochrome dyes exhibiting excitement at different wavelengths and suitable filters, this form of microscopy can be employed for the high resolution (≤ 0.2 µm) detection and localization of bacterial, fungal and other microorganisms in tissues. It is also a fruitful technique for localizing nucleic acids and specific sites of antibody-antigen reactions in structures of almost all properly prepared biological sections. There are two types of fluorescence. Primary natural fluorescence is the capacity of some substances or structures (e.g. chloroplasts, collagen fibres) to fluoresce naturally (intrinsic). Some materials that have been fixed using formaldehyde-based fixatives also show intrinsic fluorescence (e.g. red blood cells) and control slides must be used to evaluate fluorescence. Alternatively, along with control slides, material can be quenched using iron, mercury or iodine, amongst others, to destroy natural fluorescence. Secondary fluorescence is produced after the interaction of substances that are not naturally fluorescent with fluorescent dye (e.g. green fluorescent protein (GFP), fluorecein, acridine orange) that tag and specifically demonstrate tissue and or structural components in sectioned material. More sophisticated staining approaches can demonstrate up to several areas of interest concurrently.

Figure a-b. a) Fluorescently labeled bovine pulmonary artery endothelial cells. Nuclei are stained blue with DAPI, microtubules are marked green by an antibody bound to FITC and actin filaments are labeled red with phalloidin bound to TRITC; b) yeast cell membrane visualized by some membrane proteins fused with red fluorescent proteins (RFP) and GFP fluorescent markers. Imposition of light from both fluorescent labels results in yellow color. (From Wikipedia, the free encyclopedia).

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Electron Microscopes: Two basic types of instruments are called electron microscopes. Both were invented at about the same time but have fundamentally different uses. a)

Transmission electron microscopes: The transmission electron microscope (TEM) projects electrons through a thin section (60-120 nm thick) of tissue stained with metals (usually lead citrate and uranyl acetate) to produce a two-dimensional image on a phosphorescent screen. The brightness of areas within the image is proportional to the number of electrons that are transmitted through the specimen and reflect differences in specimen composition and its translucency (electron lucid and electron dense regions). Resolution of most biological material can be ≥ 2 nm which is superb and superior to that achieved using optical microscopy.

Figure. TEM image of alveolar surface cells in mammalian lung section

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Scanning electron Microscopes: The scanning electron microscope (SEM) produces an image that gives a threedimensional impression of the surface of the specimen. This microscope uses a 2 to 3 nm spot of electrons that scans the surface of the specimen to generate secondary electrons that reflect from the specimen surface and are then captured by a detector. The image is produced over time as the entire region of interest of the specimen is scanned. The image contrast (dark and light areas) can be later doctored to provide pseudo-coloration (usually green or sepia) for a more striking aesthetic appearance. Resolution of most biological material can be ≥ 2 nm which is superb and superior to that achieved using optical microscopy.

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Figure. Low magnification SEM image of an insect head showing compound eye architecture.

Figure. Pseudocolored SEM image of bone marrow cells.

c) Scanning transmission electron microscopes: A third type of electron microscope is the scanning transmission electron microscope (STEM or STM), which has features of both TEM and SEM and uses a scanning beam of electrons to penetrate thin specimens. The study of surfaces using STEM is an important part of physics, with particular applications in semiconductor physics and microelectronics. In chemistry, surface reactions also play an important part, for example in catalysis. The STEM works best with conducting materials, but it is also possible to fix organic molecules on a surface and study their structures. For example, this technique has been used in the study of DNA molecules.

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Figure. A STEM image of a salt (sodium sulphate) recrystallized from methanol.

Light microscopy allows the viewing of living and/or dead, fixed or frozen cells and tissues. Electron microscopy allows the viewing of only fixed or frozen cells but at high resolution. However, compared to the compound microscope, the electron microscope is more expensive and far less easy to transport from place to place.

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2) General Principles of Light Microscopy Parts of Microscope:

Figure showing parts of a binocular compound microscope (from blog.makezine.com/compoundmicroscope-parts.jpg)

1. Ocular lens or eyepiece: Most are 10x to 12.5x magnification. The microscopes used in this department are binocular (two eyepieces). The ocular lenses you are using are compensating eye pieces. Compensating eye pieces are corrected for the different types of aberrations common to lenses (see below). Eyepieces for binocular microscopes must be accurately paired with equal centration, magnification and field in order to reduce eye strain. Inter-ocular distance should also be adjusted and the microscopist should sit at the correct height for the eyepieces to come to the exact height of the observer’s eyes. Correct eye height can be achieved by rotating the eye-piece head 180°.

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2. Body tube: Contains mirrors and prisms which direct the image to the ocular lenses. 3. Nosepiece: holds the objective lenses, rotates and is fixed into place with a nosepiece screw. 4. Objective lenses: Microscope objectives are the most important components of a light microscope because they determine the quality of images that the microscope is capable of producing. There is a wide range of objective designs available that feature excellent optical performance and provide for the elimination of most optical aberrations (see below). There are usually 3 to 4 objectives on our microscopes, 4x, 10x, 40x, 100x oil immersion. Moreover, our microscopes have fixed position objective lenses--the stage moves up and down rather then the lens.

By convention, objective lenses are color coded and have numbers and letters written on them.

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The yellow band tells us that it is a 10X objective lens. Red is 4X, Blue is 40X or 60X and White is 100X. The first number "10" is the power (10X). The 0.25 is the Numerical Aperture. Numerical aperture as applied to microscope objectives is a measure of the ability to gather light and resolve fine specimen detail at a fixed object distance. The 160 is a standard DIN measurement in millimeters of the tube length of the microscope required for this lens to work properly. Finally, the 0.17 is the thickness in mm of the cover slip that you should use. 0.17mm cover slips correspond to a number 1 cover slip.

Some more expensive objective lenses, including some of the ones we use in this department also have the word “PHACO” or “Ph” indicating that it can be used for phase contrast microscopy. This word is followed by a number (1-3) which corresponds to the same condenser setting number on the universal condenser. A PHACO or PH code (Ph1, Ph2, Ph3) indicated on the phase contrast objective shows the size of the phase plate inside the objective. (The code is irrelevant to the magnifying power of the objective.) Lastly, the objective may also have additional information inscribed on their outside. Inscriptions may include lens corrections for spherical, chromatic and curvature aberrations, manufacturer and other specialized optical properties of the lens (see above). Microscope objectives are typically designed to be parfocal, which means that when one changes from one lens to another on a microscope, the sample stays in approximate focus. Microscope objectives are characterized by two parameters, namely, magnification 21

and numerical aperture. The former typically ranges from 5× to 100× while the latter ranges from 0.14 to 0.7, corresponding to focal lengths of about 40 to 2 mm, respectively. For high magnification applications, an oil-immersion objective or water-immersion objective has to be used. The objective is specially designed and refractive index matching oil or water must fill the air gap between the front element and the object to allow the numerical aperture to exceed 1, and hence give greater resolution at high magnification. Numerical apertures as high as NA 1.6 can be achieved with oil immersion. Immersion Media: Most low-power objectives are designed to be used "dry" with air as the imaging medium. Higher magnification objectives commonly use liquid immersion media (water, glycerin or oil) to help correct aberrations and increase numerical aperture (see below). 5. Stage: Movable platform on which slides are mounted for viewing; all of the scopes have mechanical stages with X,Y vernier scales. In the figure below, the Vernier moves up and down (Y-axis) and to the left and right (X-axis) to measure a position on the scale. They are useful in microscopy because when the position of an object on a slide is recorded using the scales, the slide can be removed and stored. Recording object position enables the microscopist to find the object again when the slide is re-inspected.

How to use a Vernier Scale: Vernier scales allows a precise reading of some value.

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The "pointer" is the line on the vernier labelled "0". In the figure on the left, the measured position is almost exactly 756 in whatever units the scale is calibrated in. If you look closely you will see that the distance between the divisions on the vernier are not the same as the divisions on the scale. The 0 line on the vernier lines up at 756 on the scale, but the 10 line on the vernier lines up at 765 on the scale. In the figure on the right, the vernier is at a different position, and the pointer, the line marked 0, does not line up exactly with one of the lines on the scale. Here the "pointer" lines up at approximately 756.5 on the scale. If you look you will see that only one line on the vernier lines up exactly with one of the lines on the scale, the 5 line. This means that the reading is 756.5. 6. Condenser: A sub-stage lens which focus the light on the specimen. Our microscopes have condensers that move up and down to focus the light beam. Microscopes may either have a single lens Abbe condenser suitable for bright field microscopy or a universal condenser for bright field, dark field and phase contrast microscopy.

A simple two-lens Abbe condenser is illustrated in the figure above. Light from the microscope illumination source passes through the condenser aperture diaphragm, located at the base of the condenser, and is concentrated by internal lenses, which then project light rays through the specimen in parallel. The size and numerical aperture (see below) of the light cone is determined by adjustment of the aperture diaphragm. After

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passing through the specimen (on the microscope slide), the light diverges into an inverted cone with the proper angle to fill the front lens of the objective.

A universal condenser is featured in the above figure. It differs considerably from the simple two lens Abbe condenser in that it is a revolving turret with up to six “ring” stops each of which can be fitted with a different type of condenser lens appropriate for different types of light microscopy and microscope objectives. The sub-stage condenser has a variety of functions. Amongst those are, it concentrates light on the object thereby regulating its visibility, it allows for the use of bright field, phase, and/or dark-field microscopy . 7. Iris Diaphragm: the diaphragm is located just below the stage and controls the amount of light which passes to the specimen and can affect image contrast and resolution. 8. Focusing knobs: Focus knobs move the stage and specimen up and down. There are two focusing knobs on microscopes. One is a coarse (inside) and the other a fine focus (outside) knob. The coarse focus is used to bring the specimen into approximate focus; the fine focus brings the specimen into sharp focus. The coarse focus knob should not be used when observing material using the 100x objective as this may damage the objective lens or break the specimen slide. 9. Light source: The scopes have built in light sources. The rheostat ON/OFF switch is located either on the scope or on the external power supply and is used to regulate light intensity.

Light, Lenses, Aberrations and Lens Corrections: 1) Light:

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Light is composed of photons and is a form of electromagnetic radiation that travels through space in straight, parallel lines as waves. The shape of a wave is given by its amplitude, phase, wavelength and frequency.

There are certain phenomena associated with light, namely interference, reflection, refraction and diffraction that are associated with microscopy. a) Interference: Two waves (of the same wavelength) are said to be in phase if the crests (and troughs) of one wave coincide with the crests (and troughs) of the other, as in the figure below.

Figure: Constructive interference

In this case the resultant wave would have twice the amplitude of the individual waves one says that constructive interference has occurred. Therefore, a combination of identical rays in similar phase relationship doubles the wave amplitude. If the crest of one wave coincides with the trough of the second, as in Fig. 22.9 they are said to be completely out of phase,.

Figure: Destructive interference

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In this case the two waves would cancel each other out - one says that destructive interference has occurred. At a point of constructive interference the net amplitude of the two waves is a maximum, whereas at a point of destructive interference, the net amplitude is a minimum or cancelled out. When light is reflected off a more dense medium with higher index of refraction, crests get reflected as troughs and troughs get reflected as crests. The wave is said undergo a 180° change of phase on reflection. The net effect of the phase change is that the reflected ray ``jumps ahead" by half a wave length. This property forms the basis of phase-contrast and differential interference microscopic optics. b) Reflected light: If a material surface is microscopically smooth and flat, such as float glass, light rays can be reflected from its surface. The angle of incoming (incident) and reflected light waves will be the same.

c) Refracted Light: Incident light is slowed down as it enters glass from air. If the light rays are not perpendicular to the glass then they will be bent. This will cause a shift in ray direction (refracted light). White light is refracted by a prism into red, orange, yellow, green, blue and violet. The shorted the wavelength the more it is refracted. Hence blue light is refracted more than red light. The degree of separation of the two ends of the spectrum is a measure of the dispersion. Different parts of the spectrum can be crowded more than others with certain lenses. Lenses with positive (convex) and negative (concave) curvature and with different dispersive characteristics can be combined as composite lenses (see below) to correct one another’s defects.

Refractive Index: The amount of refracted light bending depends on the indices of refraction of the two media and is described quantitatively by Snell's Law.

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The refractive index (RI) of air is 1.00 and for microscope glass 1.51. Therefore, light will bend considerably as it moves from air through a histological specimen mounted on a glass slide. This reduces the amount of light gathered by the objective lens which diminishes image resolution. At low and medium magnifications this does not affect image resolution noticeably. However, at higher magnifications (60 and 100x objectives) image is seriously degraded without use of immersion fluids. To reduce bending, a drop of water (RI = 1.33), glycerol (RI = 1.47) or immersion oil (RI = 1.51-1.52) is placed between the slide and objective lens surfaces to improve light capture and resolution. e) Diffracted Light: Diffraction depends on the wave nature of light. As parallel light waves pass over an edge they are bent or diffracted. Similarly, as light passes through a biconvex lens the red end of the spectrum is diffracted more than the blue end (recall that the blue end of the spectrum was refracted more).

2) Lenses a) Lens overview: Lenses used in microscopy come in a variety of shapes. They may be spherical lenses: (A), biconvex; (B), biconcave; (C), planoconvex; (D), planoconcave; (E), concavoconvex, periscopic convex, converging meniscus; (F), convexoconcave, periscopic concave, diverging meniscus; (G, H), cylindrical lenses, concave and convex (see below).

1) Biconvex (++ or converging) lenses: These lenses are thicker in the middle than at their edges. A magnifying glass is an example of a biconvex lens. Parallel light rays entering a biconvex lens will converge and focus (principle focus) on the other side. The distance between the principle focus and the centre of the lens is called the focal length (f).

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2) Biconcave (-- or diverging) lenses: These are lenses that are thicker at the edges than in the middle. When parallel light passes through the lens, the refracted light rays diverge so that they appear to come from a single focal point. The distance between the focal point and centre of lens is the focal distance. The image formed is virtual and diminished (smaller).

3) Composite lenses: Composite lenses are those in which different types of lenses (see below) are glued together to correct for aberrations (see below) associated with the passage of incident light rays through a single lens type. They are the type of lenses found in the eyepiece, objective and condenser lenses of the microscopes used in this department.

Figure: Composite lens arrangement to correct for chromatic aberration

3) Aberrations a) Spherical aberration: These artifacts occur when light waves passing through the periphery of a lens are not brought into focus with those passing through the center as illustrated in the figures below. Waves passing near the center of the lens are refracted only slightly, whereas waves passing near the periphery are refracted to a greater degree resulting in the production of different focal points along the optical axis. This is one of the most serious resolution artifacts because the image of the specimen is spread out rather than being in sharp focus.

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Although microscope manufacturers expend a considerable amount of resources to produce objectives free of spherical aberration, it is possible for the user to inadvertently introduce this artifact into a well-corrected optical system. By utilizing the wrong mounting medium (such as live tissue or cells in aqueous environments) with an oil immersion objective or by introducing similar refractive index mismatches, microscopists can often produce spherical aberration artifacts in an otherwise healthy microscope. Also, when using high magnification, high numerical aperture dry objectives, the correct thickness of the cover glass (suggested 0.17 mm = # 1 cover slip) is critical.

c) Chromatic aberration: This type of optical defect is a result of the fact that white light is composed of numerous wavelengths.

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When white light passes through a convex lens, the component wavelengths are refracted according to their frequency. Blue light is refracted to the greatest extent followed by green and red light, a phenomenon commonly referred to as dispersion. The inability of the lens to bring all of the colors into a common focus results in a slightly different image size and focal point for each predominant wavelength group. This leads to colored fringes surrounding the image as illustrated in the figure below:

Objectives which do not carry a special inscription stating otherwise are likely to be achromats. Achromats are satisfactory objectives for routine laboratory use, but since they are not corrected for all colors, a colorless specimen detail is likely to show, in white light, a pale green color at best focus (the so-called secondary spectrum). A simple achromat lens is illustrated in Figure 4 below.

The proper combination of lens thickness, curvature, refractive index, and dispersion allows the lens doublet to reduce chromatic aberration by bringing two of the wavelength 30

groups into a common focal plane. If fluorspar is introduced into the glass formulation used to fabricate the lens, then the three colors red, green, and blue can be brought into a single focal point resulting in a negligible amount of chromatic aberration. These lenses are known as apochromatic lenses and they are used to build very high-quality chromatic aberration-free microscope objectives. Modern microscopes utilize this concept and today it is common to find optical lens triplets (Figure) made with three lens elements cemented together, especially in the higher-quality objectives. For chromatic aberration correction, a typical 10x achromat microscope objective is built with two lens doublets, as illustrated in the figure below, on the left. The apochromat objective illustrated on the right in the below figure contains two lens doublets and a lens triplet for advanced correction of both chromatic and spherical aberrations.

c) Other Lens Aberrations: These include a variety of effects including astigmatism, field curvature, and comatic aberrations that are easily corrected with proper lens fabrication. i) Astigmatism: There are two distinct forms of astigmatism. The first is a third-order aberration, which occurs for objects (or parts of objects) away from the optical axis. This form of aberration occurs even when the optical system is perfectly symmetrical. This is often referred to as a "monochromatic aberration", because it occurs even for light of a single wavelength. This terminology may be misleading, however, as the amount of aberration can vary strongly with wavelength in an optical system.

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The second form of astigmatism occurs when the optical system is not symmetric about the optical axis. This may be by design (as in the case of a cylindrical lens), or due to manufacturing error in the surfaces of the components or misalignment of the components. In this case, astigmatism is observed even for rays from on-axis object points. This form of astigmatism is extremely important in ophthalmology, since the human eye often exhibits this aberration due to imperfections in the shape of the cornea or the lens. ii) Field curvature: In optical microscopy, field curvature is an optical aberration that most microscopists are familiar with. Field curvature occurs when lenses that have curved surfaces are used. Field curvature originates when visible light rays are focused through a lens that is curved. The image plane that is produced by the curved lens will be curved. Field curvature causes the specimen when observed through the eyepiece of the microscope to either appear focused in the center or on the edges. However, not both the center and the edge will be brought into focus.

iii) Comatic aberrations: Comatic aberrations are similar to spherical aberrations, but they are only encountered with off-axis objects and are most severe when the microscope is out of alignment. In this instance, the image of a point is asymmetrical, resulting in a comet-like (hence, the term coma) shape. Coma is often considered the most problematic aberration due to the asymmetry it produces in images. It is also one of the easiest aberrations to demonstrate. On a bright, sunny day, use a magnifying glass to focus an image of the sun on the sidewalk and slightly tilt the glass with respect to the principal rays from the sun. The sun's image, when projected onto the concrete, will then elongate into a comet-like shape that is characteristic of comatic aberration.

Objective Correction for Optical Aberration Objective Type Spherical Aberration

Chromatic Aberration

Field Curvature

Achromat

1 Color

2 Colors

No Yes

Plan Achromat

1 Color

2 Colors

Fluorite

2-3 Colors

2-3 Colors

No

Plan Fluorite

3-4 Colors

2-4 Colors

Yes

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Plan Apochromat

3-4 Colors

4-5 Colors

Yes

Numerical Aperture and Depth of Field a) Numerical Aperture: The numerical aperture of a microscope objective is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. Image-forming light waves pass through the specimen and enter the objective in an inverted cone as illustrated in the figure (below). A longitudinal slice of this cone of light reveals the angular aperture, a value that is determined by the focal length of the objective.

The greater the angle the greater the numerical aperture. Numerical aperture also determines the depth of field. The greater the numerical aperture the smaller the depth of field (see below).

Depth of Field: Depth of field (or depth of focus) refers to the distance over which the image is in focus. As magnification, resolution and numerical aperture are all increased the depth of field decreases.

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Depth of field can be changed somewhat by adjusting the field diaphragm of the microscope. As the aperture is closed the depth of focus and contrast increases but resolution is diminished.

Magnification and Resolution Magnification: Numerical aperture determines the resolving power of an objective, but the total resolution of a microscope system is also dependent upon the numerical aperture of the substage condenser. The higher the numerical aperture of the total system, the better the resolution. a) Magnification: Magnification is the increasing in size of an object. In microscopy magnification is the ocular (eyepiece) lens times the objective lens (e.g if objective is x 40 and the ocular lens is x10 the overall magnification is x400. The range of useful magnification for an objective/eyepiece combination is defined by the numerical aperture of the microscope optical system. There is a minimum magnification necessary for the detail present in an image to be resolved, and this value is usually rather arbitrarily set as 500 times the numerical aperture (500 x NA) and defined by the equation: Useful Magnification (total) = 500 to 1000 × NA (Objective). At the other end of the spectrum, the maximum useful magnification of an image is usually set at 1000 times the numerical aperture (1000 x NA) as given by the equation above. Magnifications higher than this value will yield no further useful information or finer resolution of image detail, will usually lead to image degradation and is considered empty magnification. The table below lists the common objective/eyepiece combinations that lie in the range of useful magnification.

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Range of Useful Magnification (500-1000 x NA of Objective) Objective Eyepieces (NA) 2.5x (0.08) 4x (0.12) 10x (0.35) 20x (0.55) 40x (0.70) 60x (0.95) 100x (1.42)

10x

12.5x

15x

20x

25x

---

---

---

x

x

---

---

x

x

x

---

x

x

x

x

x

x

x

x

x

x

x

x

x

---

x

x

x

---

---

x

x

---

---

---

b) Resolution: While there is a clear relationship between magnification and resolution, it is often assumed that magnification is what microscopists are after. However, what we actually want is objects magnified in so far as new magnification betrays new detail not seen before. This concept is known as resolution. The magnification of a newspaper picture will not bring out new detail; in fact the image becomes less clear and hence more confusing. The resolution of a microscope objective is defined as the smallest distance between two points on a specimen that can still be distinguished as two separate entities. Resolution is a somewhat subjective value in microscopy because at high magnification, an image may appear unsharp but still be resolved to the maximum ability of the objective. Resolution depends on the wavelength of light and the shorter the wavelength the better the resolution. The best resolution for a bright field microscope is using a blue filter, and is about 0.2 microns = 200 nm, or about 100,000 lines/cm. By comparison the eye can resolve 1’ (1 minute of an arc 1/60°) = 350 lines/cm at 250 mm distance owing to the spatial arrangement of cones retina. Use of fluorescent microscopy, which employs UV light, provides better resolution than blue light, owing to its shorter wavelength. The following provides insight into the relationship between magnification and resolution. In the figure below, resolved lines are seen to be separated, while those that appear to be fused are unresolved. If this figure is magnified some 10x new detail will become apparent (=useful magnification) while higher levels of magnification will betray no new detail (=empty magnification). 35

Micrometry: In addition to examining specimens for their shape and orientation, it is useful to determine their dimensions (length x width), in micrometers (µm). Accurate measurements can be achieved using an eyepiece reticule (ocular micrometer) that has been calibrated against a stage micrometer.

Ocular micrometers are calibrated by comparing the ocular micrometer scale with a calibrated stage micrometer. The stage micrometer is a microscope slide that has a carefully calibrated scale which is divided into 0.1 mm and 0.01 mm units. Therefore, the distance between the small divisions is 10 µm. In summary, the eyepiece ocular is superimposed over the stage micrometer with each objective. By simple calculation, the size of the object between a certain number of eyepiece divisions can be determined. If 10 eyepiece divisions correspond with 1 stage division, 1 eyepiece division equals 1 µm, while an object 6 eyepiece divisions in length is 6 µm. By convention, and as indicated above, the first object dimension recorded is the length followed by width. Procedure: 1. Install the 10X ocular containing the ocular micrometer disc in the microscope. 2. Place the calibrated stage micrometer slide on the stage and focus on the scale.

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3. Adjust the field so that the zero line of the ocular disc scale is exactly superimposed upon the zero line of the stage micrometer scale. 4. Without moving the stage micrometer, locate the point as far to the extreme right as possible where any two lines are exactly superimposed upon each other. 5. Count the number of divisions (mm) on the stage micrometer between the zero line and the superimposed line to the far right. 6. Count the number of ocular divisions between the zero line and the superimposed line to the far right. 7. Divide the distance determined in step 5 by the number of ocular divisions in step 6 and multiply by 1000 to give the ocular micrometer units in µm stage micrometer divisions (mm) x 1000 µm = µm per ocular unitocular micrometer divisions mm. 8. Repeat steps 3 through 7 for each objective on the microscope. If the ocular micrometer is moved to a different microscope scope, the calibration procedure must be repeated. If a new objective is added to the microscope, the calibration procedure must be done for the objective.

Path of Light through compound microscope: While there are a number of component parts to the modern compound light microscope, in the simplest terms a compound light microscope can be considered as having two lenses, (eyepiece, and objective lenses) arranged in series in a mirrored tube above a specimen stage, a condenser lens below the stage, and a light source. The path of light through these lenses is shown below.

Figure . Path of light through a simple microscope (after Druly and Wallington 1980)

The condenser lens and light source are used for contrast and illumination. The objective lens (O) magnifies the illuminated specimen SP to produce a real and inverted image S1P1 because the specimen is further away from the focal length of the objective lens. The 37

eyepiece (E) is close to the image S1P1 within the focal length of the eyepiece lens. A magnified upright and virtual image of S1P1 is produced by the eyepiece and will be seen by the observer’s eyes at S2P2. Thus the specimen will be magnified twice but inverted once for visual observation. This is why the specimen appears to move left when it is really moved to the right on the stage. There are two general forms of illumination recognized in microscopy. 1) Critical (or Nelsonian) Illumination: This method of microscope illumination was first developed by British microscopist Edward Nelson using optical principles advanced by Ernst Abbe. Nelsonian illumination relies on using the sub-stage condenser to produce a focused image of the light source in the plane of the specimen to achieve a somewhat even illumination condition over the entire view field. Light emitted from the lamp must be focused by the substage condenser so that an image of the filament is produced in the specimen plane on the slide surface. While this form of illumination is good for resolution, it produces a harsh disturbing view of the specimen. Consequently this form of microscopy has been superseded by the far more efficient Köhler method of microscope illumination.

Figure. Set up for Critical (or Nelsonian) illumination.

2) Köhler method of microscope illumination: The majority of modern light microscopes employ the Köhler method of microscope illumination. This technique is recommended by all manufacturers of modern laboratory microscopes because it can produce specimen illumination that is uniformly bright and free from glare, thus allowing the user to realize the microscope's full potential. In Köhler illumination, an image of the light source is focused at the condenser aperture diaphragm to produce parallel (and unfocused) light through the plane of the specimen or object. A magnified image of the light source below the condenser (at the aperture 38

diaphragm) produces a wide cone of illumination that is required for optimum resolution of the specimen. The size of the condenser aperture diaphragm can be used to control the numerical aperture of the light cone that illuminates the sample and reduce unwanted stray light and glare. In addition, imaging of dust and other imperfections on the glass surfaces of the condenser is minimized. Efficient sample illumination is very dependent upon proper alignment of all the optical components in the microscope, including the illumination source. The microscopist should become familiar with the adjustment range of each component and should practice aligning these with different samples and objectives.

Figure. Set up for Köhler illumination.

Procedure for setting up Köhler illumination in bright field microscopes lacking a field diaphragm: Note: Because we lack centering telescopes we are unable to centre Ph annular diaphragms. However, the annular diaphragms on the universal condenser turret in your microscope have been pre-adjusted and centered and further adjustments are unnecessary. • • • • •

Turn on the power switch to the microscope for illumination; Adjust the distance between eyepieces (interpupillary distance) to merge the left and right view fields into one; Turn the coarse focus knob to lower the stage to its lowest level; Place the specimen slide on the stage with the cover-glass facing upward. Open the claw of the specimen holder open with your finger at either the claw base or tip, then tilt and fix the slide with the claw; Rotate the revolving objective lens nosepiece to bring the 10x objective into the optical path; 39

• • •

• • •

• • •

Looking into the eyepieces slowly rotate the coarse focus knob to raise the stage. When the specimen image appears, stop rotating the coarse focus knob; Rotate the fine focus knob and precisely focus the image. Adjust the eyepiece focusing rings according to the difference between your left and right eyesight. While looking into the right eyepiece with your right eye, focus on the specimen by rotating the right eyepiece focusing ring ---- not the fine focus knob. Then repeat this for the left eyepiece. Eyepiece focusing is important as it reduces the onset of eye strain and headaches, which will result from examining specimens over long periods with improperly focused eyepieces; The condenser now needs positioning. For this swing the 10x objective into the optical path. Turn the condenser turret to setting “A”. This setting is for bright field illumination; Close the aperture diaphragm to minimum aperture by rotating the field diaphragm ring immediately below the “A” condenser setting; While looking into the eyepieces, rotate the condenser focus knob to defocus the aperture diaphragm image on the specimen surface (focus on the edge of the diaphragm image); you should see a poorly illuminated and granular image. Slowly raise the condenser with closed aperture and you will see a focus plane in which the image is bright but granular. Raise the condenser above this plane until granularity is lost. The condenser is now set for proper illumination. Open the aperture diaphragm and adjust light intensity so that it is neither blinding nor too low. This is somewhat subjective. Rotate the field diaphragm centering screws until the field diaphragm comes to the center of the view field; Switch to the 40x objective and rotate the diaphragm ring until the image just fills the view field.

. Setting Up Köhler Illumination in microscopes with field apertures: 1. Focus on the specimen. 2. Close the field diaphragm to its most closed state so that you can see the edges of the diaphragm (may be blurry) in the field of view. 3. Use the condenser focus knobs to bring the edges of the field diaphragm into the best focus possible. 4. Use the condenser-centering screws to center the image of the closed field diaphragm in the field of view. 5. Open the field diaphragm just enough so that its edges are just beyond the field of view. 6. Adjust the condenser diaphragm to introduce the proper amount of contrast into your sample. The amount of contrast added will depend on the sample, however too much contrast can introduce artifacts into your images. 7. Adjust the light intensity as necessary. To adjust light intensity, it is best to use a neutral density filter rather than increasing or reducing the supply of power to the light source. Neutral

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density filters block all wavelengths of light equally, while changing the power to the light source will alter the balance in the spectrum of incident light giving a yellow/brown appearance to the image. Bright Field Illumination Set-up:

• •

Rotate the condenser setting so that the indication A faces front (toward you); the condenser performs like an Abbe condenser described above. Objectives with a magnification higher than 4X can be used for bright field microscopy.

When using bright field illumination use of a blue filter placed above the field condenser will

improve resolution. If you are evaluating histological material stained with hematoxylin and eosin (stains tissues shades of blue and red), use of a green filter will improve contrast, especially for photomicroscopy. Phase Contrast Illumination set-up:

Aligning the Ph annular diaphragm of the condenser and the phase plate of the objective is necessary for phase contrast microscopy. Rotate the universal condenser and you will note that it is identified with the following settings: “A”, Ph1, Ph2, Ph3, and “DF”. As outlined above, setting “A” is used for bright field illumination only and uses objectives. Settings Ph1, Ph2, Ph3 correspond to settings on the 10X, 40X and 100X objectives. To use the 10X objective for phase contrast, rotate the condenser setting to Ph1 and swing the 10X objective into the path of light. Phase contrast effects cannot be obtained if different codes are used in combination. Use of a green interference filter placed above the field condenser will improve contrast during Ph microscopy. However, it will also reduce resolution. Dark-Field Illumination Set-up: Perform the following for dark-field microscopy. • • •

Rotate the condenser turret so that the DF setting faces toward you. Insert an objective having a magnification of at least 10X and a numerical aperture of no more than 0.7 into the optical path. Raise the condenser as far as it will go when viewing specimens mounted on glass slides. If the slide is too thick, the illumination may not reach the specimen. When this happens, prepare another specimen using a thinner glass slide.

Care and Maintenance: Microscopes are expensive. The student microscopes you are using are in the neighbourhood of $1500 ea. Research microscopes in this department range from $ 15,000 to $ 55,000, depending on the quality of the objectives with which they are fitted. Therefore you take good care of your microscope.

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Filter and lens cleaning: Do not get dust, fingerprints etc on lenses or filters as this will adversely affect the image. If any of the lenses or filters get dirty, clean them as follows: • • •

Use an air blower to blow away dust. If that does not work brush away the dust with a soft brush or wipe it away gently with gauze; If there are fingerprints or grease on a lens or filter, dampen a piece of soft clean cotton cloth, lens paper or gauze with absolute alcohol (ethyl or methyl) and gently wipe. Do not use the same piece of cloth etc. to wipe more than once. Use petroleum benzine to clean off immersion oil. Wipe with ethyl or methyl alcohol after the oil has been removed finishes the clean up process. Do not use solvents such as xylene to clean objective lenses as it will erode the lens cement and can damage the lens.

Storage: Store the microscope under conditions of low humidity where mold is not likely to grow. Some molds will grow on lens coatings, produce glass etching acids and subsequently damage lenses. Cover the microscope with a vinyl dust cover to protect it from dust. Ensure that the lamp housing is cool to the touch before putting on the dust cover.

References: Bozzola, JJ and LD Russell. 1992. Electron Microscopy: Principles and techniques for biologists. Jones and Bartlett Publishers, Boston Drury, RAB and EA Wallington.1980. Carleton’s Histological Technique. Oxford University Press, Toronto. Davies, P. 1980. Quantitative morphology of the Lung: A practical manual. Harvard Medical School. Boston. http://www.microscopyu.com/articles/formulas/formulasna.html http://en.wikipedia.org/wiki/Differential_interference_contrast_microscopy http://en.wikipedia.org/wiki/Fluorescence_microscope http://en.wikipedia.org/wiki/Optical_microscope http://en.wikipedia.org/wiki/Dark_field_microscopy http://en.wikipedia.org/wiki/Phase_contrast_microscopy http://en.wikipedia.org/wiki/K%C3%B6hler_illumination http://en.wikipedia.org/wiki/Astigmatism http://www.microscopyu.com/articles/formulas/formulasna.html

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http://www.olympusmicro.com/primer/anatomy/kohler.html http://www.olympusmicro.com/primer/anatomy/illumination.html http://www.olympusmicro.com/primer/anatomy/objectives.html http://en.wikipedia.org/wiki/Snell's_law simple.wikipedia.org/wiki/Wave_(physics)

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Appendix 1. Figure showing preparation of wet mount specimens.

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Appendix 2. Quantitative Microscopy. Appendix 3. Photomicroscopy

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