Microscopy

The history of microscopy is closely linked to the history of microbiology. Early microscopists, including Hooke, Divini, Kircher, and van Leeuwenhoek, were among the first individuals to describe microscopic life forms . Robert Hooke, in his landmark 1665 book Micrographica, included many illustrations of microscopic forms. In observation XVIII, Hooke first used the term "cell" to describe the microscopic architecture of thin pieces of cork. Hooke's Micrographica was a best seller of its time, inspiring many scientists to discover the microbiological world. In his 1678 letters to the Royal Society in London, Antony van Leeuwenhoek provided detailed descriptions of protozoa. His descriptions of "very small animalcules" included drawings of basic organism shapes and descriptions of their movement. In an effort to observe the microscopic world in ever greater detail, van Leeuwenhoek built simple microscopes of increasing power. Some of these microscopes were capable of x200 magnification. Van Leeuwenhoek's 1683 letters to the Royal Society included the first ever description of living bacteria from dental plaque sampled from between his teeth. Microscopy continued to play an important role in the study of biology and medicine, and almost two centuries later, the only scientific equipment Charles Darwin took with him on the voyage of the Beagle was a simple microscope. Today, light microscopy is used not only in biological sciences such as microbiology, botany, forensics, pathology, and cell biology but also in metallurgy, engraving, chemistry, mineralogy, gemology, computer chip design, and microsurgical applications. This chapter will attempt to describe the basic concepts of light microscopy as they are practiced in the microbiology laboratory.

 

TECHNICAL BACKGROUND AND DEFINITION OF TERMS

Aberration

Aberrations are unwanted artifacts in the microscopic image that are caused by elements in the optical path. Aberration can be caused by physical objects such as dust or oils on the optical surfaces, by alterations in the light path caused by improper alignment or aperture settings, and by imperfections in the lens systems. Two main types of optical aberrations can occur when white light passes through a convex lens: spherical aberration and chromatic aberration. Spherical aberration is hallmarked by images that appear to be in focus in the center of the field and out of focus at the periphery. Chromatic aberration occurs because shorter light wavelengths are refracted to a greater extent than longer wavelengths. This wavelength separation (also called dispersion) produces color fringes within the image field. Chromatic aberration can be reduced or eliminated in optical systems by combining two lenses with different color dispersion characteristics.

Contrast

Contrast is a measure of the differences in image luminance that provides gray scale or color information. Contrast is expressed as the ratio of the difference in luminance between two points divided by the average luminance in the field under optimum conditions, the human eye can detect the presence of 2% contrast

Depth of Field and Depth of Focus

Depth of field is a subjective measure of the vertical distance between the nearest and farthest objects in the specimen that appear to be in sharp focus. Depth of field decreases as the numerical aperture (NA) of the lens increases Depth of focus is the area around the image plane where the image will appear to be sharply focused. The image plane is formed within the microscope tube at or near the level of the ocular lenses. Microscopes with greater depth of focus allow the user to employ ocular lenses with different working distances, magnification factors, and visual compensation systems without losing image sharpness. Like depth of field, depth of focus depends upon the NA of the objective. However, depth of focus increases as the NA increases.

Immersion Fluid (Immersion Oil) Immersion fluid is a term used to describe any liquid that occupies the space between the object and microscope objective lens. Immersion fluids are usually required for objectives that have working distances of 3 mm or less. Many microscopy applications employ immersion fluids that possess the same refractive index as the glass slide (refractive index 1.515). This procedure produces a homogeneous optical path which minimizes light refraction and maximizes the effective NA of the objective lens. Immersion fluids are also used between the condenser and the microscope slide in transmitted light fluorescence microscopy and in dark-field microscopy to minimize refraction, increase the NA of the objective, and improve optical resolution.

Koehler Illumination

Kohler illumination was first introduced in 1893 by August Kohler of the Carl Zeiss Corporation as a method of providing the optimum specimen illumination In this procedure, the collector lens projects an enlarged and focused image of the lamp filament onto the plane of the aperture diaphragm. Because the light source is not focused at the specimen, the specimen is bathed in a uniformly bright, glare-free light that is not seriously affected by dust and imperfections on the glass surfaces of the condenser. Koehler illumination is required to produce maximum optical resolution and high-quality photomicrographs.

Mechanical Tube Length

Mechanical tube length describes the light path distance within the microscope body tube. Tube length is measured from the objective opening in the nosepiece to the top edge of the observation tube. Tube length is usually inscribed on the barrel of the objective as the length in millimeters (e.g., 160, 170, 210, etc.) for fixed lengths or the infinity symbol (co) for infinity-corrected tube lengths. Many of the newer objectives are infinity corrected, while older objectives will be corrected for 160-mm (Nikon, Olympus, and Zeiss) or 170-mm (Leica) tube lengths Differences in mechanical tube length are among the major reasons why objectives from one manufacturer cannot be used on a different

Numerical Aperture

NA is a measure of the r gilt-gathering capability of a lens or condenser. Higher-NA objectives have better resolving power and brighter images than lower-NA objectives.

Higher-NA objectives also have shallower depths of field. The equation for determining NA is given by NA n, sine, where n is the refractive index of the imaging medium between the objective and the specimen and E is one-half the angular aperture of the objective (HL2) (3, 5, g, 15). For visible light microscopy using standard immersion oils, the theoretical maximum for NA is about 1.52. In practice, however, a lens cannot accept an 1800 cone of light, so B has to be slightly less. This results in a maximum practical NA of about 1.40.

Refractive Index (Index of Refraction)

The index of refraction is the ratio of the velocity of light in a vacuum to its velocity in a transparent or translucent medium. As the refractive index of a material increases, light beams entering or leaving a material are deflected (refracted) to a greater extent. The refractive index of a medium depends upon the wavelength of light passing through it. Light beams containing multiple wavelengths (e.g., white light) are dispersed when they move into a different medium because each wavelength in the beam is refracted to a slightly different degree. Light dispersion causes chromatic aberration in microscope objectives Refractive index is also an important variable in calculating NA (see "Numerical Aperture" above). Moving from a high-dry microscope objective that uses air as the imaging medium (refractive index of air 1.003) to an oil immersion objective of the same power (refractive index of immersion oil 1.515) increases the maximum theoretical NA of a given lens from 1.0 to 1.5, producing a 50% increase in light-gathering capability

Resolution (Resolving Power)

The resolution of an optical microscope is defined as the shortest distance between two points that can be distinguished by the observer or camera system as separate entities. The resolving power of a microscope is the most important feature of the optical system because it defines our ability to distinguish fine details in a specimen. The theoretical limit of resolution for a given lens is defined mathematically as r K/(2NA), where r is the resolution, K is the imaging wavelength, and NA is the numerical aperture of the lens. From this equation, it is obvious that only the light wavelength and NA directly affect the resolving power. Thus, a 40x oil objective with an NA of 1.30 can have the same resolving power as a 100x oil 0b objective. In the same manner, the resolving power of a 100x oil objective is higher when using UV wavelengths than it is when using visible light.

Working distance

Working distance is the distance between the objective front lens and the top of the cover glass when the specimen is in focus.The working distance of an objective generally decreases as magnification increases The working distance of an objective may not be inscribed on the barrel of older objectives, but newer objectives often contain the working distance in millimeters. Longer-working-distance objectives are important when examining the inside surface of glass tubes (tube cultures) and cell culture flasks.

SIMPLE MICROSCOPES

Common objects such as jewelers' loupes, handheld photographic slide viewers, and simple magnifying or reading glasses are all examples of simple microscopes in routine use today. A simple microscope is composed of a single biconvex magnifying lens which is thicker in the center than at the periphery. In contrast with compound microscopes, simple microscopes produce a magnified image that is in the same orientation as the original object. Because of their low NA, simple microscopes have limited resolution and magnifying power. Most commercial magnifiers are able to produce an x2 to x30 magnification, and the better lenses will have a resolution of about 10 um. Simple magnifiers are useful for dissection, examination of bacterial colonies, and interpretation of agglutination reactions.

COMPOUND MICROSCOPES

The first compound microscopes were constructed around 1590 by Dutch spectacle makers, Zaccharias Janssen and Hans Janssen. The Janssen microscope consisted of an object lens (objective) that was placed close to the specimen and the eye, or ocular, lens that was placed close to the eye. The lenses were separated by a body tube. In this microscope, the objective lens projected a magnified image into the body tube and the eyepiece magnified the projected image, thereby producing a two-stage magnification. Modern compound microscopes still use this general design and have two separate lens systems mounted at opposite ends of a body tube.

The stereoscopic microscope combines two compound microscopes which produce separate images for each eye. Stereoscopic microscopes may have one or two separate objectives, and many have a zoom magnification function. These microscopes are used for reflected or transmitted illumination, but the absence of a substage condenser limits their NA and resolution. Stereomicroscopes are useful in examining colonial morphology of bacteria, fungi, and cell cultures.

The modern light microscope is composed of optical and mechanical components that, together with the mounted specimen, make up the optical train. The optical train of a typical bright-field microscope consists of an illuminator (light source and collector lens), a substage condenser, specimen, objective, eyepiece, and a detector. The detector can be a camera or the observer's eye.

Specimen illumination is one of the most critical elements in optical microscopy. Inadequate or improper sample illumination can reduce contrast in the specimen and significantly decrease the resolving power of any microscope. There are numerous commercially available illuminators for microscopes, but 50- or 100-watt tungsten halogen lamp systems are frequently used due to their low cost and long life. Light generated by the light source is passed through a collector and a field lens (Fig. 3) before being directed into the substage condenser and onto the specimen. Image-forming light rays are captured by the microscope objective and passed into the eyepieces or a camera port. Alignment of the optical components of a microscope is critical to produce a good image.

The field diaphragm

The field diaphragm is located in the light path between the light source and the substage condenser. This iris- like mechanism controls the width of the light beam that enters the substage condenser. The field diaphragm does not affect the optical resolution, NA, or intensity of Illumination. However, the field diaphragm should be centered in the optical path and opened far enough that it just overfills the field of view. This adjustment is important for preventing glare and loss of contrast in the observed image. When the field diaphragm is opened too far, scattered light and reflections can degrade image quality.

Substage Condenser

The substage condenser is typically mounted beneath the microscope stage in a bracket that can be raised or lowered independently of the stage. The substage condenser gathers light from the field diaphragm and concentrates it into a cone of light that illuminates the specimen with uniform intensity over the entire field of view.

Adjustment of the substage condenser is probably the most critical element for achieving proper illumination, and it is the main source of image degradation and poor quality photomicrography. The condenser light cone must be properly adjusted to optimize the intensity and angle of light entering the objective. Because each objective has different light-gathering capabilities (NA), the substage condenser should be adjusted to provide a light cone that matches the NA of the new objective. This is done by adjusting the aperture (or condenser) diaphragm control. Substage condensers on newer microscopes have a scale embossed on the condenser and an index mark on the aperture control that allows the user to quickly switch from one NA range to another. Many manufacturers are now synchronizing the NA gradations to correspond with the approximate NA of the objectives.

In clinical laboratory practice, the condenser aperture is often made smaller to improve the contrast of wet mounts and some stained preparations. The condenser is sometimes moved downward for the same purpose. These practices are effective for a few applications, but they will result in decreased optical resolution. Specimen illumination intensity should not be adjusted by opening and closing the condenser diaphragm or by moving the condenser laterally in the light path. Illumination intensity should be controlled through the use of neutral density filters placed into the light path or by reducing the amp voltage.

Reducing the voltage, however, will also alter the color of the incoming light, and voltage changes are not recommended for photomicroscopy.

Objectives

The objective lens is the most important single determinant of the quality of the image produced by a particular microscope. When choosing a microscope, the purchaser must select the magnification factor, NA, and the level of correction for each objective. Lenses with higher NA values will have higher resolution and produce a brighter field of view. The level of optical correction in the objective will depend upon the ultimate use of the microscope.

Achromatic (achromat) objectives are the least expensive objectives found on laboratory microscopes. Achromat objectives are corrected for axial chromatic aberration in two wavelengths (red and blue), and they are corrected for spherical aberration in one color (green). The limited correction of achromatic objectives can cause a number of optical artifacts when specimens are examined and photographed in color (e.g., green images often have a reddish-magenta halo). Achromat objectives produce the best results with light passed through a green filter and when performing black and white photomicroscopy.

Flatness of field is also a problem when using straight achromat objectives because the center of the field is in focus while the edges are out of focus. In the past few years, most manufacturers have begun providing flat-field corrections for achromat objectives.

These objectives are called plan-achromats.

The next higher level of correction and cost is found in objectives called fluorites or semiapochromats. Fluorite objectives are produced from advanced glass formulations that allow for greatly improved correction of optical aberration. Like the achromat objectives, the fluorite objectives are corrected chromatically for red and blue light. unlike achromats, fluorites are corrected spherically for two or three colors instead of a single color.

The superior correction of fluorite objectives compared to achromats enables these objectives to be made with a higher NA. Fluorite lenses therefore produce brighter images than achromats. Fluorite objectives have better resolving power than achromats and provide a higher degree of contrast, making them better suited for color photomicrography in white light.

Apochromats

Apochromats are the most highly corrected microscope lenses and the most costly. Apochromats are corrected chromatically for three colors (red, green, and blue), almost eliminating chromatic aberration, and are corrected spherically for either two or three wavelengths. Apochromat objectives are the best choice for color photomicrography in white light. Because of their high level of correction, apochromat objectives usually have, for a given magnification, higher NAs than do achromats or fluorites.

Fluorescence Objectives

Fluorescence objectives are designed with quartz and other special glasses that have high transmission rates for IJV, visible, and infrared light. These objectives are extremely low in autofluorescence, and they use specialized optical cements and antireflection coatings that protect the lens and allow it to operate with a wide variety of excitation wavelengths.

Correction for optical aberration and NA values in UV fluor objectives usually approaches that of apochromats, which contributes to image brightness and enhanced image resolution.

The primary drawback of high-performance fluorescence objectives is that many are not corrected for field curvature and produce images that do not have uniform focus throughout the entire field of view. This is not a large problem when performing direct or indirect fluorescent antibody testing, but it can be troublesome if you have to use the same objectives for bright-field or phase-contrast microscopy. Microscope objectives that use air as the medium between the coverslip and the objective lens are considered dry objectives. The maximum working NA of a dry objective system is limited to 0.95. Higher NA values can only be achieved using optics designed for immersion media. Immersion media have the same refractive index and dispersion values as glass (refractive index — 1.51). The use of immersion media produces a homogeneous light path from the coverslip to the lens so that light is not refracted away from the objective.

The use of immersion fluids and immersion lenses significantly increases the NA and the optical resolution of the system. In addition to "oil" lenses, specially corrected objective lenses designed for glycerin and water immersion are commercially available. The proper immersion fluid type is always stamped on the side of the objective. The advantages of oil immersion objectives are severely compromised if the wrong immersion fluid is utilized. Microscope manufacturers produce immersion objectives with tight refractive index and dispersion tolerances.

It is therefore advisable to use only the immersion fluid recommended by the

objective manufacturer. Mixing of immersion fluids from different manufacturers should be avoided because mixing can produce unexpected crystallization artifacts or phase separations that compromise image quality. Many high-power (NA, 20.8) dry objectives are engineered to operate through 0.17-mm coverslips (designated as number 1%). In practice, however, the total thickness of the specimen-coverslip sandwich can be greater or less than 0.17 mm due to variations in coverslip and/or mounting fluid thickness .

Under these conditions, there will be noticeable spherical aberration in the microscopic image. A 0.2-mm deviation in coverslip thickness will produce an 8% decrease in image intensity when using a 0.79 NA objective and 57% with a 0.85 NA high-dry objective. Therefore, some of the more

advanced dry objectives are engineered with a coverslip correction collar that adjusts the objective lens elements to compensate for coverslip thickness variations. Objectives with a coverslip correction collar are labeled Corr, w/Corr, or CR. However, this labeling is usually unnecessary because the objective has a distinctive knurled ring and graduated scale on the side. The expected coverslip thickness for an objective is etched on the barrel of the objective.

Eyepiece or Ocular Objective

The eyepiece or ocular objective contains the final lens system in the optical train. The purpose of the ocular objective is to magnify and focus the projected image onto the eye of the viewer. Ocular lenses generally have a magnification factor of x 10 to x20, and the total magnification of the microscope is the product of the objective magnification and the ocular magnification. Thus, a microscope with a 40x objective and a lox ocular lens have a magnification value of x400. Many eyepieces have a shelf at the level of the fixed eyepiece diaphragm that allows for the insertion of ocular micrometers, pointers, or crosshairs. This shelf is located at the focal plane of the image projected by the objective lens so that the inserted element is in focus when the specimen image is in focus.

DARK-FIELD MICROSCOPY

Dark-field microscopy is a specialized illumination technique that is used in the clinical laboratory to detect thin organisms such as spirochetes and Leptospira. High-resolution dark-field microscopy utilizes a specialized high-NA cardioid dark-field condenser that blocks the central light path light and produces a hollow cone of illumination that is directed away from the objective lens at an oblique angle. Bacteria on the slide have a slightly different refractive index than the surrounding medium, and light rays passing through the organism are refracted into the objective lens. Light rays that do not pass through an organism do not enter the lens. This type of illumination produces bright organism profiles against a dark background. Dark-field microscopy requires careful alignment of the condenser and the placement of immersion oil between the slide and the sub stage condenser. Dark-field microscopy, when done correctly, increases the resolution of the microscope to 0.1 um or less. The resolution of bright-field microscopy is 0.2 u.

PHASE-CONTRAST MICROSCOPY

In the early 1930s, the Dutch physicist F. Zernike introduced a new method for testing optical systems which he called phase contrast. Originally designed as a macroscopic testing system, the use of phase-contrast techniques in microscopic systems allowed the user to examine unstained biological specimens that were virtually transparent when observed under bright-field illumination. Zernike was awarded the Nobel Prize in 1953 for his contributions to microscopy.

Phase contrast is essentially an interference method wherein a ring annulus is placed directly under the lower lens of the condenser to produce a hollow cylinder of light. This light is essentially unchanged as it passes into the objective, and it arrives at the rear focal plane of the objective in the shape of a ring. Light that goes through the specimen is refracted and slowed slightly so that it is out of phase by about one-quarter wavelength from the unchanged light. This out-of-phase light is spread over the entire focal plane. Light passing through the rear focal plane of the objective interacts with a separate ring-shaped phase plate that alters the direct light path by another one-quarter wavelength when the direct light and the refracted light arrive at the image plane; they are out of phase by one-half wavelength. This out-of-phase light interacts destructively so that specimen details appear as dark areas against a lighter background.

Because the phase-shifting calculations are based on a one-quarter wavelength of green light, the phase image has the best resolution when a green filter is placed in the light path. Green filters also allow the microscopist to use less-expensive achromat lenses that are spherically corrected for green light. Phase microscopy is an important tool for examining living and/or unstained material in wet mounts

and cell cultures.

However, phase-contrast microscopy has lower resolution than bright-field microscopy of stained specimens In addition, viewed objects are often surrounded by halos that can obscure boundary details. Phase-contrast microscopy does not work well with thick specimens because the phase shift may be greater than the expected one-quarter wavelength. In phase-contrast microscopy, structures within living cells appear as hills or craters, depending upon their optical thickness. This pseudo-three-dimensional effect can greatly enhance image contrast in unstained specimens.

FLUORESCENCE MICROSCOPY

The fluorescence microscope was developed in the early 1900s, and many of the initial microscopic studies involved identification and localization of compounds that autofluoresced when irradiated with UV light. In the lg30s, a number of investigators began using fluorescent compounds to identify specific tissue components and infectious agents that did not autofluoresce These stains are not organism specific, but rather, they bind to and stain specific structures within the organism. Examples of this type of staining include acridine orange, auramine-rhodamine, calcofluor white, Evans blue, and Hoechst 33258. The use of fluorochrome-antibody conjugates (immunofluorescence) was first described in the lg40s when Coons et al used fluorescein-labeled antibodies to detect pneumococcal polysaccharide antigens in tissue sections of infected mice.

Fluorescent antibody staining expanded significantly with the development of fluorescein isocyanate in 1950 (13) and the more stable fluorescein isothiocyanate (FITC) derivative in 1958. Today, fluorescence microscopy is also used in conjunction with nucleic acid hybridization to visualize the location of fluorescent in situ hybridization and multicolor fluorescent in situ hybridization probes .

Fluorescence microscopy is dependent upon the ability of fluorescent substances to absorb light of a certain wavelength and emit light at a longer wavelength. Fluorescence microscopes used for clinical microbiology most often utilize fluorochromes that absorb near- UV light energy and reemit that light at a lower, green or yellow wavelength. To work properly, the fluorescence microscope must irradiate the specimen with I-IV excitation ight and separate the much weaker emitted light from the brighter excitation light so that only the emitted light reaches the eye. The resulting image consists of brightly shining areas against a dark background. Older fluorescent microscopes are configured for dark-field illumination. In these instruments, LIV excitation light enters a dark-field condenser and the light is directed onto the specimen at an oblique angle. Fluorescent compounds in the specimen absorb the excitation light, and the emitted light is collected by the objective lens.

The emitted light then passes through a barrier filter to remove any excitation light that may enter the objective. These microscopes are difficult to use for routine diagnostics because the condenser and the objective must be carefully oiled. The dark-field condenser also reduces the effective NA of the objectives, thereby producing dim images that lack resolution. Most modern fluorescence microscopes use reflected light (epifluorescence). In these instruments, the excitation light is directed downward through the objective and onto the specimen. The emitted light and the reflected excitation light are collected by the objective, and they pass through a dichoric mirror which removes the excitation light and allows the longer- wavelength emitted light to form an image.

With epifluorescence, the objective acts as a condenser and the alignment and oiling issues associated with a dark- field condenser are eliminated The visual field is brighter with epifluorescence, the resolution is higher, and fluorescence quenching only occurs in the field of view. Fluorescence microscopy requires high levels of illumination because the quantum yield of most fluorochromes is low. The most common lamps are mercury vapor (HBO) lamps, ranging in wattage from 50 to 200 watts, or xenon vapor (XBO) lamps, which range from 75

to 200 watts. It should be noted that lamp wattage is not necessarily a measure of usable brightness in a fluorescence lamp. The HBO 100-watt lamp is 4 times brighter than the 200- watt HBO lamp and 11 times brighter than the XBO 150-watt lamp. When purchasing a fluorescence microscope, it is also important to determine whether the emission spectrum of the lamp is compatible with the fluorochromes used in the laboratory.

 HBO and XBO lamps are under high pressure, and care must be taken to prevent the lamps from exploding. One should never touch these lamps with bare hands because oils on the fingers can etch or discolor the glass. Fluorochromes must be excited by specific light wavelengths to generate the maximum amount of emitted light. Therefore, specific exciter and barrier filter combinations are used to maximize the quantum yield of the fluorophore. Exciter filters are used to select the required light wavelengths from the spectrum of light generated by the amp Excitation filters are provided in narrow, medium, and wide band-pass configurations that pass a narrower and wider range of light frequencies, respectively. Barrier filters block shorter light wavelengths and allow longer wavelengths to pass through the filter. Barrier filters are important because they remove the high-intensity excitation light that could overwhelm the low-intensity emitted l' get. Barrier filters also prevent I-IV light from entering the eye where it can cause cataracts and retinal damage.

Wide band-pass barrier filters general y produce brighter images, but care must be taken to prevent the introduction of background right that could overwhelm the emitted light. Epifluorescence microscopes also have a dichromatic mirror (beam splitter) that reflects the incoming excitation light to the objective and allows the emitted light to pass to the barrier filter and onto the objectives. In most modern epifiuorescence microscopes, the barrier filter, excitation mirror, and beam splitter are housed in removable optical blocks, and several of these blocks can be installed in the microscope at one time. This configuration allows the user to quickly change the excitation and barrier filters to accommodate different fluorochromes. Care must be exercised when selecting optical blocks. The excitation filter should match the excitation wavelength of the fluorophore, and the emission barrier should allow the emitted light to pass through. For example, direct fluorescent antibody testing for viral antigens in cell smears typically employs FITC-labeled antibodies and an Evans blue counterstain. Choosing an optical block with a 450- to 490-nm-pore-size excitation filter and a 515-nm-pore-size wide band-pass barrier filter will produce a bright field of view, and the counterstained cells will appear orange-red. By selecting a more restricted band-pass barrier filter (520- to 560-nm pore size), the field of view will be darker and the red emitted light from the Evans blue counterstain will not be visible. The images produced by optical block will have more contrast because the background is darker. Both filter combinations are appropriate for this task, but the final choice will depend upon user preference.

One of the major problems in the use and examination of fluorescent microscopic images is the tendency of fluorophores to lose fluorescence when exposed to excitation light for several minutes. This loss of fluorescence is caused by two mechanisms: photobleaching and quenching. Photobleaching (fading) is a permanent loss of fluorescence that is caused by chemical damage to the fluorophore. Quenching is caused by the presence of free radicals, salts of heave,' metals, or halogen compounds (2). Quench'ng can also be caused by transfer of emission light energy to other fluorescent molecules in close proximity to the fluorophore in a process called fluorescent resonance energy transfer. To lessen the effect of Quenching, slides should be stored in the dark at 2 to BOC.

 In addition, the user should block the excitation light path when not viewing or photographing the specimen. Most epifiuorescence microscopes have a shutter in the light path for this purpose. Quenching can be a significant problem when photographing fluorescent images because the shutter may be open for a minute or more. Quenching can be reduced somewhat by the addition of free radical scavengers such as p-phenylenediamine, 1,4-diazabicyclo(2,2,2)-octane (DABCO) (25), orn-propylgallate  to the mounting fluid. p-phenylenediamine and n- propy gallate can be used to reduce quenching in FITC and rhodamine. DABCO is slightly less effective than p-phenylenediamine for FITC fluorescence, but unlike p-phenylenediamine, DABCO does not darken when exposed to ight and it is safer to use. Quench-resistant mounting media are also available from Vector Laboratories (Burlingame, CA), Molecular Probes, Inc. (Carlsbad, CA), and Bio-Rad Laboratories (Hercules, CA).

LINEAR MEASUREMENTS (MICROMETRY)

The first reported micrometric procedures were credited to Antony van Leeuwenhoek, who used fine grains of sand as a gauge to determine the size of human erythrocytes. Since then, a variety of methods have been used to determine the dimensions of microscopic organisms. The crudest method for determining size in the clinical laboratory involves comparing the object size to the measured or calculated •Wew field size. Other micrometric techniques include the addition of polystyrene beads of known size into the specimen.Comparative measurements are then performed utilizing a photomicrograph or digital image. The accuracy of this method is variable and depends on the homogeneity of the comparison objects. Direct measurement of microorganisms can be done by placing them on calibrated microscope slides or counting chambers. The accuracy of this method depends on the separation distance between ruled lines but averages between 10 and 50 micrometers. The most common procedure used in the clinical laboratory,' utilizes a graduated scale (reticle) located within one of the eyepieces. Reticles must be calibrated against a stage micrometer for each objective. The accuracy of this type of measurement is

approximately 2 to 10 micrometers (3 to 5%), depending on magnification and the resolution of the stage micrometer.

PHOTOMICROSCOPY

Microscopists began capturing microscopic images on film shortly after the photographic process was invented (4). Micrographic images have long been used for investigations of morphology, in scientific publications and lectures, and in teaching. Modern film technologies have high resolution and clarity, but the use of photomicrographs in day-to-day microscopy has been hampered by long turnaround times associated with film development and printing. Reacquiring fluorescence images is a particular concern because the fluorescence can fade (Z).

The availability of high-quality digital cameras has significantly changed how photomicrographs are used in the microbiology laboratory. Today, it is not unusual for digital photomicrographs to be shared with experts via the Internet. This process significantly extends the capabilities of the on-site microbiologist and can enhance patient care. Microscope-based digital cameras and video systems are also used to perform "plate rounds in remote hospitals and clinics within a multihospital system.

Newer Internet technologies involving robotic microscopes and high-resolution video systems now allow microbiologists to change the focus and change slide positioning of a microscope located anywhere in the world and view the resulting images on a monitor in their office. The availability of digital photomicroscopy has significantly enhanced the microbial identification process, and it has helped to standardize microbe identification. A wide variety of microscopes are currently available that have integrated camera systems and sophisticated light metering and exposure controls. Accessory cameras are also available from a large number of aftermarket manufacturers.

 The performance and optical characteristics of these systems are too diverse to discuss in a single chapter, and the camera specifications necessary for digital microscopy will depend upon the type of images that will be captured. Light sensitivity is a key element in any digital camera, and camera

Manufacturers utilize the film-equivalent International Organization for Standardization (ISO) numbers to rate the light sensitivity of image sensors in digital cameras. ISO numbers range from 80 to 3200 and the higher the ISO value, the more sensitive the image sensor. Some digital cameras have manual or automatic sensitive adjustments that can alter the image sensor so that the camera can be used in low-light conditions. Unfortunately, this increased sensitivity is usually achieved by amplifying the signal from the image sensor. This type of

Signal amplification also increases the background levels and decreases image quality. The "heart" of the digital camera is the image sensor, a silicon chip that measures and captures light. Presently, there are two types of image sensors, the complementary metal- oxide semiconductor (CMOS) and the charge- coupled device (CCD) sensor. Consumer cameras typically use CMOS chips because they are easier to manufacture. Many CMOS cameras can be used to capture images generated during bright-field microscopy. CMOS cameras tend to be smaller and less expensive than CCD cameras. In addition, CMOS cameras use less power, and they have faster frame rates, fewer artifacts (smear and blooming) caused by charge transfer between adjacent pixels, and the ability to include "higher-level" camera functions such as image stabilization and wireless control . The major disadvantage of the CMOS camera is its lower light sensitivity.

CCD and slow-scan CCD cameras, which were first developed for astronomy, are the current cameras of choice for low-light-level fluorescence microscopy. However, these cameras are much more expensive than CMOS cameras. Photographs are a stern judge of microscopic quality, and the purchase of an expensive camera system does not automatically confer the ability to produce high-quality images. Optics, optical train alignment, and proper illumination are the most important factors in the acquisition of high-quality photomicrographs. Optical image deficiencies as evidenced by chromatic variance and poor image clarity are more noticeable when using a digital camera than when using 35-mm film. Color photography can be especially demanding because specimens may appear yellow or blue under tungsten halogen (3,200-K color temperature) light depending upon whether the lamp voltage is above or below the recommended g-volt setting. Photographs will also appear blue if the daylight blue filter is not removed from the light path. Photographs can also appear yellow when tungsten halogen (3,200-K) illumination is used in conjunction with daylight (5,500-K) film or digital cameras designed for daylight photography. Under these conditions, a Kodak 80A (3,200-K to 5,500- K) color conversion filter should be placed in the light path to achieve the proper 5,500-K color temperature.

Not all microbiologists can afford a microscope with an integrated camera system. Simple eyepiece cameras can also be used to capture bright-field images. The simplest configuration for eyepiece photography involves the use of a point-and-shoot digital camera. Some improvisation may be necessary with this method because few adapters are available for coupling a fixed-lens camera to the microscope eyepiece. Instead, the microscopist can use a camera tripod or some other support bracket to hold the camera in its proper position. Entry of stray light can be minimized by using a piece of black polyvinyl chloride (PVC) pipe with an appropriate diameter and a black camera cloth. During photography, the camera lens system should be set to infinity focus (the default in fixed lens cameras), and the lens should be positioned so that it is at the eye point (focal point) of the eyepiece. The location of the eyepoint can be determined by holding a piece of white paper just above the objective with the microscope turned on and focused A bright circle of light will be projected onto the paper. The eyepoint is the position where the light circle is smallest. Photographs produced under these conditions are often acceptable, but they may be dark. Cameras with adjustable aperture settings should be set to the largest apefture value (smallest f-stop number) to maximize the amount of light entering the camera. This method will also produce some chromatic aberration (due to different lens correction factors) and vignetting (pipe view effect).

 Another method for photomicroscopy is to use the camera port on microscopes fitted with a trinocular head. Olympus and Nikon have introduced adapters that allow their digital cameras to attach to the camera tube of their microscopes. In addition, camera tube and eyepiece adaptors for a number of digital cameras are available from Microscope Depot (Tracy, CA). Photography under these conditions is best done using a camera with through- the-lens exposure metering. These devices work well if the exposure is no longer than several seconds or shorter than one-third of a second Many of these cameras have built- in flashes that should be turned off during photomicroscopy. These cameras may have problems with fluorescence microscopy due to the extreme contrast of fluorescent images and the tendency of metering systems to average exposure values over the entire field.

CARE AND USE OF THE MICROSCOPE

Proper care and maintenance of the microscope will prolong the usable life of the instrument and allow for more accurate interpretation of microbiological images. The microscope should be kept in a low-vibration, low-dust environment to facilitate viewing and decrease damage to the optical systems. The optical elements should be kept completely free of dust, dirt, oil, solvents, and any other contaminants. Ideally, the microscope should be covered and the lamp should be turned off when the microscope is not in use.

Do not touch the optical surfaces with your fingers. Keep the lenses clean and be sure to remove oil or mounting fluid from the objectives, condenser, and mechanical stage after each session. Avoid dragging the high-dry objective through oil or fluorescence mounting fluid. One way to avoid accidental contact with these fluids is to place the high-dry objective and the oil immersion objective in the nosepiece on opposite sides of the owl-power objective Lenses should be dusted with residue-free compressed air and cleaned with lens paper and a commercial lens cleaner that is approved by the microscope manufacturer. Organic solvents such as alcohols and acetone should not be used on the lenses because the solvent may dissolve the optical mounting cement. Unused spaces in the nosepiece should be plugged and the eyepieces should remain installed at all times to prevent introduction of dust into the body tube. The stage should be cleaned regularly, and any spilled immersion oil must be removed or slides will stick when they are moved across the stage. Spilled immersion oil also collects dust and grit that can damage the optical and mechanical parts. Microscopists should not attempt to remcwe or dissemble the objectives, as this increases the potential for damage.

This is a job that is best left to professionals. The gears and rackwork should be cleaned and treated with new grease at intervals specified by the manufacturer. Do not use light oil on the gears or bearing surfaces because this may cause the condenser and stage to sink from their own weight. Periodic cleaning and adjustment by a professional microscope repair person also help to extend the usable life of the microscope.

ERGONOMICS

Peering into a microscope eyepiece for long periods is not an activity for which the body is well adapted. Microscope work requires the head and arms to be locked in a forward position and inclined toward the microscope with rounded shoulders. This unusual positioning is further exaggerated when the feet are placed on the ring-style footrests that are common to many laboratory stools.

 Poor posture and awkward positioning during microscopy can cause pain or injury to the neck, wrists, back, shoulders, and arms. In one regional survey of cytotechnologists, Kalavar and Hunting found that 70.5% of respondents reported neck, shoulder, or upper back pain during microscopy and 56% had an increased prevalence of hand or wrist symptoms. Eyestrain and leg and foot discomfort have also been documented with long-term microscope use. When using older microscopes, users often have their heads inclined up to 450 from vertical and their upper backs may be inclined by as much as 300. Even 300 inclinations of the head can produce significant muscle contractions, fatigue, and pain For this reason, microscopists should be taught to sit upright and hold their heads in neutral positions (3Z)• During microscopy, the laboratorian should sit erect and maintain the natural curve of the spine. The lower back and shoulder blades should be supported by the chair, and a lumbar support cushion should be used if necessary. The legs and feet should rest firmly on the floor or a footrest. The chair should have a pneumatic height adjustment, and the seat should have a sloping front edge to prevent undue pressure on the thighs. The backrest should be adjustable for both height and angle, and the chair should have a five-pointed star base with caster wheels. Knee spaces, which are often used for laboratory storage, should be free from obstructions, and there should be a minimum of 2 inches of clearance between the thigh and the bottom of the desk or counter. Obstructions that prevent the microscopist from holding his or her shoulders perpendicular to the ocular axis of themicroscope should be removed. The upper arms should be perpendicular to the floor, with the elbows close to the body. The forearms should be parallel with the floor, and the wrists should be straight.

The head should be upright, and the neck should bend as little as possible, preferably no more than 10 to 150. The eyepieces should be just below the eyes, and the eyes should 00k downward at a 30 to 450 angle. The use of tilting microscope heads can significantly improve the comfort of the microscopist. Repetitive motions of the hands and the contact stress of arms resting on (the edge of) a hard surface can cause pain and nerve injury, leading to repetitive stress injuries and/or carpal tunnel syndrome. The use of padded arm rests can moderate some of these problems. In addition, to prevent stiffening of the muscles during microscopy, microscopes should not be placed under an air vent.

Most laboratory microscopes are used by multiple individuals, and it is often a challenge to find conditions or microscope configurations that satisfy everyone. Some laboratories place microscopes on books or heavy,' blocks of wood to accommodate taller microscopists. This configuration creates a number of problems. If the microscope is raised to a sufficient height to prevent neck flexion, users may be forced to bend their wrists into an unnatural position. If the microscope is lowered to allow the forearms to remain parallel to the floor, the neck is forced to bend. Lowering the chair to its lowest position causes leg discomfort.

Individuals of short stature may have to raise the chair to a level where their feet no longer touch the floor. Foot rests can ameliorate this problem, but some individuals may have insufficient space under the bench top to accommodate their legs. In practice, most laboratories will elect to use a suboptimum, but workable, microscope configuration that all users can employ. Under these conditions, microscopists can reduce stress and fatigue by taking 1-minute "microbreaks" every 10 to 15 minutes where they can stand, stretch, and allow the eyes to focus at a distance. More expensive solutions, including the use of ergonomic microscope tables that can be raised and lowered electrically have been employed in some laboratories. Eye fatigue can be a major problem for microscope users, especially if they have poor vision. The diopter adjustment provided on most microscope eyepieces can be adjusted to compensate for minor near- and farsightedness, thereby allowing users to remove their glasses during microscope use. The diopter adjustments do not adjust for astigmatism, and users with moderate to severe astigmatism should wear glasses when using the microscope. Most microscope manufacturers now produce high-eyepoint eyepieces that move the visual observation point further from the eyepiece, thereby facilitating the use of glasses during microscopy. Ensuring that the microscope images are as bright, sharp, and crisp as possible will also help to reduce eye fatigue and associated headaches. The importance of proper arrangement of the microscope and optical components cannot be overstressed. Proper optical arrangement and the use of newer objectives with higher NA values will produce brighter images and better resolution, which eases the strain of searching for tiny specimen details.

The use of a neutral blue (day light) filter during bright-field microscopy can also help to lessen eyestrain when examining microbiological specimens. In the future, many new microscopes will display the specimen image on a computer monitor. This innovation could alleviate many of the eyestrain problems that develop during extended microscope use.

Microscopes are as different as the people who use them, and the previous comments should not be construed as a prescription for alleviating strain or repetitive motion injuries in every situation. When purchasing a microscope, every effort should be made to allow microscopists to evaluate the new microscope under their normal working conditions. Some microscopes will be comfortable for some users and uncomfortable for others. In the long run, the feel and fit of the microscope are just as important as the optical characteristics.

CONCLUSION

Advances in the design, resolution, and ergonomics of modern microscopes have greatly enhanced our ability to study and identify microorganisms. Microscopy still has a central role in the detection of infectious agents despite high y publicized advances in DNA and RNA detection systems. Microscopic examination of clinical specimens provides a rapid and inexpensive "first pass" in the detection and identification of infectious agents. Thus, clinical microscopy will continue to be a core competency in clinical microbiology laboratories for the foreseeable future.