Microscopy Light Microscopes Image capture Magnification calculation

Basic Concepts in Microscopy

Modern compound microscopes operate using a dual stage magnifying design that incorporates a primary imaging lens, the objective, coupled to a secondary visualizing lens system known as the eyepiece orocular mounted at the opposite ends of a body tube. The objective is responsible for primary image formation at varying magnifications, while the eyepiece is used to observe the image created by the objective. Advanced microscopes feature infinity optical systems that project a parallel bundle of wavefronts from the objective rear aperture to a tube or telan lens, which in turn focuses the image at the intermediate image plane in the eyepieces. The microscopist is able to observe a greatly enlarged virtual image of the specimen by peering through the eyepieces. Magnification is determined by multiplying the individual values of the objective and eyepiece. Resolution and contrast in optical microscopy are derived through a number of optical strategies and is strongly coupled to the types of reagents used to prepare the specimen. This section discusses the basic concepts necessary for a complete understanding of microscopy, including objectives, eyepieces, condensers, magnification, numerical aperture, resolution, contrast, and optical aberrations, along with a wide spectrum of additional considerations. Review Articles

Introduction to Microscopy - Microscopes are specialized optical instruments designed to produce magnified images of specimens that are too small to be seen with the naked eye. In addition to complex designs featuring objectives and condensers, microscopes also consists of very simple single-lens instruments that are often hand-held, such as a common magnifying glass.

How the Microscope Forms Images - Optical microscopes belong to a class of instruments that are said to be diffraction limited, meaning that resolution is determined in part by the number of diffraction orders created by the specimen that can be successfully captured by the objective and imaged by the optical system.

Numerical Aperture and Resolution - The numerical aperture of a microscope objective is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance. Resolution is determined by the number of diffracted wavefront orders captured by the objective.

The Point Spread Function - The ideal point spread function (PSF) is the three-dimensional diffraction pattern of light emitted from an infinitely small point source in the specimen and transmitted to the image plane of a microscope (or other diffraction-limited optical instrument) through a high numerical aperture (NA) objective or lens system.

Illumination and the Microscope Optical Train - The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. Illumination of the specimen is the most important controllable variable in achieving high-quality images in microscopy and digital imaging.

Khler Illumination - Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography. Khler illumination was first introduced in 1893 by August Khler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination.

Microscope Optical Systems - Microscope objectives are perhaps the most important components of an optical microscope because they are responsible for primary image formation and play a central role in determining the quality of images that the microscope is capable of producing. Other components include the illumination collector, condenser, and eyepieces.

Microscope Objectives - The most important component of an optical microscope is the microscope objective. Objectives are responsible for primary image formation and play a central role in establishing the quality of images that the microscope is capable of producing.

Enhancing Contrast in Optical Microscopy - The contrast-enhancing techniques for transmitted light microscopy described in this section represent a variety of methods in sample preparation as well as optical tricks that generate intensity changes which are useful for observation and imaging.

Fluorescence Microscopy - Because of the highly sensitive emission profiles, spatial resolution, and high specificity with regards to signal-to-noise and contrast, fluorescence microscopy is rapidly becoming an important tool in genetics and cell biology, and remains at the forefront of biomedical research with the continuous introduction of new techniques.

Reflected Light Microscopy - Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers using conventional contrast-enhancing techniques.

Contrast Modes in Reflected Light Microscopy - In its standard configuration, a typical reflected light microscope is readily equipped to examine amplitude (absorption) specimens using brightfield incident light. Through the addition of auxiliary components, a variety of contrast-enhancing mechanisms can be introduced.

Understanding the Digital Image - This discussion is intended to aid in understanding the basics of light detection, the fundamental properties of digital images, and the criteria relevant to selecting a suitable detector for specific applications.

Practical Use of the Microscope - If certain simple guidelines are followed, it will be a short matter of time before a beginner is able to obtain an image of high quality. In fact, you may be surprised at how easy it is to set up the microscope correctly so that it will produce beautiful, sharp images.

Microscopy in Everyday Use - Although conventional microscope design has not necessarily been a problem for short-term use, long-term sessions have in the past created problems for scientists and technicians who used the instruments. Ergonomics is concerned with finding a better fit between people microscopes.

Care and Maintenance of the Microscope - Microscopes often represent a significant investment of funds and are sophisticated optical instruments that require periodic maintenance and cleaning to guarantee successful microscopy and perfect images.

Microscopy: Historical Perspective - For many centuries, the construction of microscopes and the underpinning optical systems was entirely an issue of exterior cosmetic craftsmanship, with the design of optical components lagging seriously behind advances in the fabrication of microscope bodies and frames. Interactive Tutorials

Optical Pathways in the Transmitted Light Microscope - The design of an optical microscope must ensure that the light rays are organized and precisely guided through the instrument. This interactive tutorial explores the function of the field and condenser aperture diaphragms of a transmitted light microscope.

Microscope Alignment for Khler Illumination - Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography or digital imaging. This interactive tutorial explores how to establish Khler illumination on a transmitted light microscope.

Objective Specifications - Microscope objectives are precision optical systems that feature a wide range of magnifications, numerical aperture, immersion media, specialized contrast applications, and other properties. This interactive tutorial examines the specifications found on typical objectives.

The Concept of Magnification - A simple microscope or magnifying glass (lens) produces an image of the specimen upon which the microscope or magnifying glass is focused. This interactive tutorial explores how a simple magnifying lens operates to create a virtual image of the specimen on the retina of the human eye.

Microscope Conjugate Planes - The conjugate planes critical for establishing proper illumination in the microscope are examined in this interactive tutorial. Four conjugate planes can be brought simultaneously into focus: the field diaphragm, the specimen plane, the intermediate image plane (where the reticule is positioned), and the human eye.

Fixed Tube Length Microscope Conjugate Field Planes - The geometrical relationship between image planes in the traditional fixed tube length (usually 160 millimeters) optical microscope is explored in this tutorial. In most of the imaging steps in the microscope optical train, the image is real and inverted, but a virtual image is also produced in one of the image planes.

Infinity Corrected Microscope Conjugate Field Planes - A majority of modern research microscopes are equipped with infinity-corrected objectives that no longer project the intermediate image directly into the intermediate image plane. Light emerging from these objectives is instead focused to infinity, and a second lens, known as a tube lens, forms the image at its focal plane.

Infinity Optical System Basics - Infinity-corrected microscope optical systems are designed to enable the insertion of auxiliary optical devices into the optical pathway between the objective and eyepieces without introducing spherical aberration, requiring focus corrections, or creating other image problems.

Field Iris Diaphragm Function - When the microscope is properly configured for Khler illumination, the field diaphragm is imaged in the same conjugate plane as the specimen, and in fact, all of the image-forming conjugate planes are simultaneously imaged into each other and can collectively be observed while examining a specimen in the eyepieces.

Numerical Aperture and Light Cone Geometry - The light-gathering ability of a microscope objective is expressed in terms of the numerical aperture, which is a measure of the number of highly diffracted imageforming light rays captured by the objective. This interactive tutorial explores the effect of numerical aperture on light cone geometry.

Airy Disk Formation - When an image is formed in the focused image plane of an optical microscope, every point in the specimen is represented by an Airy diffraction pattern having a finite spread. This interactive tutorial explores the origin of Airy diffraction patterns formed by the rear aperture of the microscope objective and observed at the intermediate image plane.

Spatial Frequency and Image Resolution - When a line grating is imaged in the microscope, a series of conoscopic images representing the condenser iris opening can be seen at the objective rear focal plane. This tutorial explores the relationship between the distance separating these iris opening images and the periodic spacing (spatial frequency) of lines in the grating.

Conoscopic Images of Periodic Structures - This tutorial explores the reciprocal relationship between line spacings in a periodic grid (simulating a specimen) and the separation of the conoscopic image at the objective aperture plane. When the line grating has broad periodic spacings, several images of the condenser iris aperture appear in the objective rear focal plane.

Numerical Aperture and Image Resolution - The image formed by an objective at the intermediate image plane of a microscope is a diffraction pattern produced by spherical waves exiting the rear aperture and converging on the focal point. This tutorial explores the effects of objective numerical aperture on the size of Airy disk patterns.

Fundamental Aspects of Airy Disk Patterns - This tutorial explores how Airy disk pattern size changes with objective numerical aperture and the wavelength of illumination. It also simulates the close approach of two Airy patterns as they approach the Rayleigh criterion for determining the ability to resolve two closely spaced objects in the microscope.

Oil Immersion and Refractive Index - One way of increasing the optical resolving power of the microscope is to use immersion liquids between the front lens of the objective and the cover slip. This tutorial explores how changes in the refractive index of the imaging medium can affect how light rays are captured by the objective, which has an arbitrarily fixed angular aperture of 65 degrees.

Condenser Numerical Aperture - The size and numerical aperture of the light cone emitted by a substage condenser is determined by adjustment of the aperture diaphragm. This interactive tutorial examines how changing the aperture iris diaphragm opening size alters the size and angle of the light cone.

Condenser Aperture Diaphragm Function - The size and numerical aperture of the light cone produced by the condenser is determined by adjustment of the aperture diaphragm. Appropriate use of the adjustable aperture iris diaphragm (incorporated into the condenser or just below it) is of significant importance in securing correct illumination, contrast, and depth of field.

Condenser Light Cones - It is critical that the condenser light cone be properly adjusted to optimize the intensity and angle of light entering the objective front lens. Each time the objective is changed, a corresponding adjustment must be performed on the condenser to provide the proper light cone to match the numerical aperture of the new objective.

Coverslip Thickness Correction - High magnification dry objectives are often subject to aberration artifacts due to variations in cover glass thickness and dispersion. This tutorial demonstrates how internal lens elements in such an objective may be adjusted to correct for these fluctuations.

Focus Depth and Spherical Aberration - Explore the three-dimensional aspects of spherical aberration that is generated when imaging deep into specimens using the meridional section of a point spread function with this interactive tutorial. Spherical aberration is a significant problem when imaging specimens in aqueous media.

Inverted Microscope Lightpaths - Microscopes featuring an inverted-style frame are designed primarily for live-cell imaging applications and are capable of producing fluorescence illumination through an episcopic and optical pathway. This interactive tutorial explores illumination pathways in the Zeiss Axio Observer researchlevel inverted tissue culture microscope.

Reflected Light Microscope Optical Pathways - Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 micrometers. Reference Library

Basic Principles in Optical Microscopy - A list of the best textbooks that provide a general knowledge of the principles and practice of optical microscopy, as well as an introduction to contrast-enhancing techniques. The volumes listed here are useful in both the classroom and the research laboratory.

Microscope Optical Systems - The microscope optical train typically consists of an illuminator (including the light source and collector lens), a condenser, specimen, objective, eyepiece, and detector, which is either some form of camera or the observer's eye. These components are often supplemented with contrast-enhancing optical elements.

Specimen Contrast - Control of image contrast in a microscope optical system is dependent upon several factors, primarily the setting of aperture diaphragms, degree of aberration in the optical system, the optical contrast system employed, the type of specimen, and the optical detector.

Phase Contrast Microscopy - Phase contrast was introduced in the 1930's for testing of telescope mirrors, and was adapted by the Carl Zeiss laboratories into a commercial microscope for the first time several years later. The technique provides an excellent method of improving contrast in unstained biological specimens.

Differential Interference Contrast (DIC) Microscopy - Differential interference contrast converts gradients in specimen optical path length into amplitude differences that can be visualized as improved contrast in the resulting image. Images produced in DIC microscopy have a distinctive shadow-cast appearance

Fluorescence Microscopy - The application of fluorescence illumination and detection in optical microscopy has ushered in a wide range of advanced applications for live-cell imaging and in vivoobservations. The articles tabulated in this section discuss the basic aspects of fluorescence, microscope configuration, fluorescent probes, software, light sources, detectors, objectives, filter sets, and a variety of other pertinent topics.

Polarized Light Microscopy - The polarized light microscope is designed to observe specimens that are visible due to their birefringent character. Polarizing microscopes must be equipped with apolarizer, positioned in the light path before the specimen, and an analyzer placed in the optical pathway between the objective rear aperture and the observation tubes or camera port.

Microscope Ergonomics - Although conventional microscope design has not necessarily been a problem for short-term use, long-term sessions have in the past created problems for scientists and technicians who used the instruments. Microscope operators often must assume an unusual and challenging position.

Introduction The numerical aperture of a microscope objective is the measure of its ability to gather light and to resolve fine specimen detail while working at a fixed object (or specimen) distance. Image-forming light waves pass through the specimen and enter the objective in an inverted cone as illustrated in Figure 1(a). White light consists of a wide spectrum of electromagnetic waves, the period lengths of which range between 400 and 700 nanometers. As a reference, it is important to know that 1 millimeter equals 1000 micrometers and 1 micrometer equals 1000 nanometers. Light of green color has a wavelength range centered at 550 nanometers, which corresponds to 0.55 micrometers. If small objects (such as a typical stained specimen mounted on a microscope slide) are viewed through the microscope, the light incident on these minute objects is diffracted so that it deviates from the original direction (Figure 1(a)). The smaller the object, the more pronounced the diffraction of incident light rays will be. Higher values of numerical aperture permit increasingly oblique rays to enter the objective front lens, which produces a more highly resolved image and allows smaller structures to be visualized with higher clarity. Illustrated in Figure 1(a) is a simple microscope system consisting of an objective and specimen being illuminated by a collimated light beam, which would be the case if no condenser was used. Light diffracted by the specimen is presented as an inverted cone of halfangle (), which represents the limits of light that can enter the objective. In order to increase the effective aperture and resolving power of the microscope, a condenser (Figure 1(b)) is added to generate a ray cone on the illumination side of the specimen. This enables the objective to gather light rays that are the result of larger diffraction angles, increasing the resolution of the microscope system. The sum of the aperture angles of the objective and the condenser is referred to as the working aperture. If the condenser aperture angle matches the objective, maximum resolution is obtained.

In order to enable two objectives to be compared and to obtain a quantitative handle on resolution, the numerical aperture, or the measure of the solid angle covered by an objective is defined as: Numerical Aperture (NA) = sin()(1) where equals one-half of the objective's opening angle and is the refractive index of the immersion medium used between the objective and the cover slip protecting the specimen ( = 1 for air; = 1.51 for oil or glass). By examining Equation (1), it is apparent that the refractive index is the limiting factor in achieving numerical apertures greater than 1.0. Therefore, in order to obtain higher working numerical apertures, the refractive index of the medium between the front lens of the objective and the specimen cover slip must be increased. The highest angular aperture obtainable with a standard microscope objective would theoretically be 180 degrees, resulting in a value of 90 degrees for the half-angle used in the numerical aperture equation. The sine of 90 degrees is equal to one, which suggests that numerical aperture is limited not only by the angular aperture, but also by the imaging medium refractive index. Practically, aperture angles exceeding 70 to 80 degrees are found only in the highest-performance objectives that typically cost thousands of dollars. The resolution of an optical microscope is defined as the smallest distance between two points on a specimen that can still be distinguished as two separate entities. Resolution is directly related to the useful magnification of the microscope and the perception limit of specimen detail, though it is a somewhat subjective value in microscopy because at high magnification, an image may appear out of focus but still be resolved to the maximum ability of the objective and assisting optical components. Due to the wave nature of light and the diffraction associated with these phenomena, the resolution of a microscope objective is determined by the angle of light waves that are able to enter the front lens and the instrument is therefore said to be diffraction limited. This limit is purely theoretical, but even a theoretically ideal objective without any imaging errors has a finite resolution. Observers will miss fine nuances in the image if the objective projects details onto the intermediate image plane that are smaller than the resolving power of the human eye (a situation that is typical at low magnifications and high numerical apertures). The phenomenon of empty magnification will occur if an image is enlarged beyond the physical resolving power of the images. For these reasons, the useful magnification to the observer should be optimally above 500 times the numerical aperture of the objective, but not higher than 1,000 times the numerical aperture.

Immersion Media One way of increasing the optical resolving power of the microscope is to use immersion liquids between the front lens of the objective and the cover slip. Most objectives in the magnification range between 60x and 100x (and higher) are designed for use with immersion oil. Good results have been obtained with oil that has a refractive index of n = 1.51, which has been precisely matched to the refractive index of glass. All reflections on the path from the object to the objective are eliminated in this way. If this trick were not used, reflection would always cause a loss of light in the cover slip or on the front lens in the case of large angles (Figure 2).

The useful numerical aperture of the objective and therefore the resolving power would be reduced by the reflection described above. The numerical aperture of an objective is also dependent, to a certain degree, upon the amount of correction for optical aberration. Highly corrected objectives tend to have much larger numerical apertures for the respective magnification as illustrated in Table 1. Objective Numerical Aperture versus Optical Correction
Magnification 0.5x 1x 2x 4x 10x 20x 40x 40x (oil) 63x 63x (oil) 100x (oil) Plan Achromat (NA) 0.025 0.04 0.06 0.10 0.25 0.40 0.65 n/a 0.75 n/a 1.25 Plan Fluorite (NA) n/a n/a 0.08 0.13 0.30 0.50 0.75 1.30 0.85 1.30 1.30 Plan Apochromat (NA) n/a n/a 0.10 0.20 0.45 0.75 0.95 1.40 0.95 1.40 1.40

Table 1

The Airy Disk and Microscope Resolution When light from the various points of a specimen passes through the objective and is reconstituted as an image, the various points of the specimen appear in the image as small patterns (not points) known as Airy patterns. This phenomenon is caused by diffraction or scattering of the light as it passes through the minute parts and spaces in the specimen and the circular rear aperture of the objective. The limit up to which two small objects are still seen as separate entities is used as a measure of the resolving power of a microscope. The distance where this limit is reached is known as the effective resolution of the microscope and is denoted as d0. The resolution is a value that can be derived theoretically given the optical parameters of the instrument and the average wavelength of illumination. It is important, first of all, to know that the objective and tube lens do not image a point in the object (for example, a minute hole in a metal foil) as a bright disk with sharply defined edges, but as a slightly blurred spot surrounded by diffraction rings, called Airy disks (see Figure 3(a)). Three-dimensional representations of the diffraction pattern near the intermediate image plane are known as the point-spread function (Figure 3(b)). An Airy disk is the region enclosed by the first minimum of the airy pattern and contains approximately 84 percent of the luminous energy, as depicted in Figure 3(c). The point-spread function is a three-dimensional representation of the Airy disk.

Resolution can be calculated according to the famous formula introduced by Ernst Abbe in the late 19th Century, and represents a measure of the image sharpness of a light microscope: Resolutionx,y = / 2[ sin()](2) Resolutionz = 2 / [ sin()]2(3) where is the wavelength of light, represents the refractive index of the imaging medium as described above, and the combined term sin() is known as the objective numerical aperture (NA). Objectives commonly used in microscopy have a numerical aperture that is less than 1.5, restricting the term inEquations (2) and (3) to less than 70 degrees (although new high-performance objectives closely approach this limit). Therefore, the theoretical resolution limit at the shortest practical wavelength

(approximately 400 nanometers) is around 150 nanometers in the lateral dimension and approaching 400 nanometers in the axial dimension when using an objective having a numerical aperture of 1.40. Thus, structures that lie closer than this distance cannot be resolved in the lateral plane using a microscope. Due to the central significance of the interrelationship between the refractive index of the imaging medium and the angular aperture of the objective, Abbe introduced the concept of numerical aperture during the course of explaining microscope resolution. The diffraction rings in the Airy disk are caused by the limiting function of the objective aperture such that the objective acts as a hole, behind which diffraction rings are found. The higher the aperture of the objective and of the condenser, the smaller d0 will be. Thus, the higher the numerical aperture of the total system, the better the resolution. One of the several equations related to the original Abbe formula that have been derived to express the relationship between numerical aperture, wavelength, and resolution is: Resolutionx,y or d0 = 1.22 / [NAObj + NACon](4) Where is the imaging wavelength of light, NACon is the condenser numerical aperture, and NAObj equals the objective numerical aperture. The factor 1.22 has been taken from the calculation for the case illustrated in Figure 4 for the close approach of two Airy disks where the intensity profiles have been superimposed. If the two image points are far away from each other, they are easy to recognize as separate objects. However, when the distance between the Airy disks is increasingly reduced, a limit point is reached when the principal maximum of the second Airy disk coincides with the first minimum of the first Airy disk. The superimposed profiles display two brightness maxima that are separated by a valley. The intensity in the valley is reduced by approximately 20 percent compared with the two maxima. This is just sufficient for the human eye to see two separate points, a limit that is referred to as the Rayleigh criterion.

A comparison may help to make this easier to understand. It is most unlikely that a telephone cable would be used for the electronic transfer of the delicate sound of a violin, since the bandwidth of this medium is very restricted. Much better results are obtained if high-quality microphones and amplifiers are used, the frequency range of which is identical to the human range of hearing. In music, information is contained in the medium sound frequencies; however, the fine nuances of sound are contained in the high overtones. In the microscope, the subtleties of a structure are coded into the diffracted light. If you want to see them in the imaging space behind the objective, you must make sure that they are first gathered by the objective. This becomes easier with a higher aperture angle and thus an increased numerical aperture. The numerical aperture of objectives increases with the magnification up to about 40x (see Tables 1 and 2), but levels off between 1.30 and 1.40 (depending upon the degree of aberration correction) for oil immersion versions. Presented in Table 2 are the calculated values for the resolution of objectives typically used in research and teaching laboratories. The point-to-point resolution at the specimen, d0, is listed in the table

along with the magnified size of the image (D0) in the intermediate eyepiece plane (using green light of 550 nanometer wavelength). Also in the table, the value n represents the number of resolved pixels if they are organized in a linear array along the field diameter of 20 millimeters (20 millimeters/D 0). Resolution for Selected Objectives
Objective/NA 0.5x / 0.15 10x / 0.30 20x / 0.50 40x / 0.75 40x / 1.30 (oil) 63x / 1.40 (oil) 100x / 1.30 (oil) d0 (m) 2.2 1.1 0.7 0.45 0.26 0.24 0.26 D0 (m) 11.2 11.2 13.4 17.9 10.3 15.1 25.8 n 1786 1786 1493 1117 1942 1325 775

Table 2 You should not try to increase the overall magnification of a microscope by using eyepieces providing a high additional magnification (for example, 16x, 20x or 25x) or other optical after-burners if the objective does not supply enough pixels at a low numerical aperture. On the other hand, you will miss subtle nuances if the objective projects very fine details onto the intermediate image, and you are using an eyepiece with a low magnification. In order to observe fine specimen detail in the optical microscope, the minute features present in the specimen must be of sufficient contrast and project an intermediate image at an angle that is somewhat larger than the angular resolving power of the human eye. As previously mentioned, the overall combined magnification (objective and eyepiece) of a microscope should be higher than 500x, but less than 1000x the objective aperture. This value is known as the range of the useful magnification. In day-to-day routine observations, many microscopists do not attempt to achieve the highest image resolution that is possible with their equipment. The resolving power of a microscope is the most important feature of the optical system and influences the ability to distinguish between fine details of a particular specimen. The primary factor in determining resolution is the objective numerical aperture, but resolution is also dependent upon the type of specimen, coherence of illumination, degree of aberration correction, and other factors such as contrast-enhancing methodology either in the optical system of the microscope or in the specimen itself. Practical Hints to Increase Resolving Power Modern microscope objectives permit the theoretical resolving power to be realized in practice provided that suitable specimens are being observed. However, there are several hints that can be followed to ensure success. These are listed below.

Are the objective and specimen clean? A fingerprint on the front lens of an air objective may be sufficient to affect the high-contrast image reproduction of a specimen as a result of unwanted scattered light. The same caveats apply to immersion objectives that are soiled with residues of resin or emulsions (such as oil and water). In these cases, careful cleaning of the objective front lens element using a soft cloth and lens cleaner or pure ethanol should alleviate problems.

Do the cover slips have the correct thickness? It is of critical importance that cover slips matched to objectives of high aperture (greater than 0.65) have the standard thickness of 170 micrometers due to the fact that cover slip thickness is taken into consideration when the objectives are designed. Therefore, if a cover slip of different thickness (less than 165 or more than 175 micrometers) is used, the quality of the optical image visibly suffers. In general, the rule of thumb for objectives having a numerical aperture of 0.7 or higher is that they can tolerate a variation of 10 micrometers, whereas lower numerical aperture objectives (0.3 to 0.7) can tolerate a higher level of deviation, usually up to 30 micrometers.

Are you using the correct immersion oil? All objectives having a numerical aperture greater than 0.95 are designed for use with immersion media (usually oil of refractive index 1.515). Immersion oil is free of polychlorinated biphenyls and exhibits hardly any autofluorescence. The image will be markedly impaired if air bubbles are introduced into the immersion layer, so oil must be carefully applied to avoid bubbles. Air bubbles can be readily visualized by removing the eyepiece and examining the objective rear focal plane through the microscope observation tubes (or using a Bertrand lens). If air bubbles are observed, the objective and specimen slide should be cleaned and the oil carefully re-applied. Illumination of the specimen is the most important variable in achieving high-quality images in microscopy and critical photomicrography or digital imaging. Khlerillumination was first introduced in 1893 by August Khler of the Carl Zeiss corporation as a method of providing the optimum specimen illumination. The manufacturers have designed modern microscopes so that the collector lens and any other optical components built into the base of the microscope will project an enlarged and focused image of the lamp filament onto the plane of the aperture diaphragm of a properly positioned substage condenser. Closing or opening the condenser diaphragm controls the angle of the light rays emerging from the condenser and reaching the specimen from all azimuths. Because the light source is not focused at the level of the specimen, the light at specimen level is essentially grainless and extended, and does not suffer deterioration from dust and imperfections on the glass surfaces of the condenser. Opening and closing of the condenser aperture diaphragm controls the angle of the light cone reaching the specimen. The setting of the condenser's aperture diaphragm, along with the aperture of the objective, determines the realized numerical aperture of the microscope system. As the condenser diaphragm is opened, the working numerical aperture of the microscope increases, resulting in greater resolving power and light transmittance. Parallel light rays that pass through and illuminate the specimen are brought to focus at the back focal plane of the objective, where the image of the variable condenser aperture diaphragm and the image of the light source will be seen in focus.

Proper configuration of the microscope in regards to illumination is possibly one of the most misunderstood concepts in optical microscopy, and is a critical parameter that must be fulfilled in order to achieve optimum performance. The intensity and wavelength spectrum of light emitted by the illumination source is of major importance, but even more imperative is that light emitted from various locations on the lamp filament be collected and focused at the plane of the condenser aperture diaphragm. The conjugate field and aperture planes critical for establishing proper illumination in the microscope are illustrated in Figure 1. In this section, the guidelines are described for establishing Khler illumination (used in transmitted and reflected light microscopy). Successful observation and imaging should occur when this procedure is executed correctly. The first steps necessary to prepare the microscope for Khler illumination involve basic procedures necessary to observe a specimen under brightfield conditions. First, if the condenser contains phase annulus rings, differential interference contrast prisms, or other masks and filters, rotate the turret to thebrightfield aperture, which should be open and not house a filter or prism. Second, choose a suitable specimen. The best specimens consist of well-stained brightfield samples, such as very thin (8 micrometer or less) animal or plant tissue. If a prepared slide is not available, a small section of 35-millimeter film (such as a transparency or negative) about 10 x 10 millimeters in size can be taped or glued to a clean glass microscope slide and flattened with a cover slip.

Steps in Establishing Khler Illumination

Switch on the light source and hold a small sheet of paper directly above the luminous field diaphragm collector lens in the base of the microscope (Figure 2(a)). The purpose of this exercise is to check whether the light source is functioning and will project light into the microscope. In cases where no light is observed and the paper remains dark, check the line cord, illuminator, and perhaps the fuse in the power unit to ensure that the electrical components are intact. If not, change the illuminator lamp. When the microscope is operating normally, a small spot of light (Figure 2(a)) will be visible on the paper.

Next, open the field diaphragm to its widest position (fully open) by turning the lever or knob. The spot of light projected onto the paper sheet (Figure 2(b)) will achieve its maximum diameter.

The next step is to move the sheet of paper and place it between the specimen and the objective (Figure 3(a)) and fully open the condenser aperture diaphragm (usually controlled by a lever or knurled knob located on the condenser housing. As the condenser diaphragm is slowly opened, the intensity of light projected onto the paper sheet will increase, achieving its maximum brightness at the fully opened setting. When using a low numerical aperture condenser equipped with a swing-in front lens element, remove the swing-lens from the light path before attempting this procedure.

The condenser height can be adjusted using the drive knob that translates this component up and down along the microscope optical axis (Figure 3(b)). Set the condenser height so that the front lens is approximately 1 to 3 millimeters beneath the lower surface of the specimen slide. Make certain the condenser front lens does not contact the slide (or push it up away from the stage). In extreme cases, driving the condenser into the specimen will result in the sample being ejected from the stage when it slips free from the retaining guide. Modern microscopes are often equipped with an adjustable stop screw that allows the top position of the condenser to be fixed at a pre-determined height.

Light should now be discernable in the microscope eyepieces (Figure 4(a)). If the light is too bright (often bright enough to cause discomfort), reduce the intensity until you find a level that is comfortable to work with. Next, set the interpupillary distance via the folding bridge of the binocular tubes. This can be done by squeezing the tubes together to decrease the distance or pulling the tubes apart to increase the separation between them. The correct distance is reached when a single large circle of light (instead of two) can be easily visualized through the eyepieces. The next step is to check to see if the microscope is equipped with focusing eyepieces. If so, turn the movable eyepiece to the zero setting. Eyeglass wearers can leave their glasses on.

Peer into the microscope and carefully move the stage, including the specimen, up and down until you see the image details as sharply as possible (Figure 4(b)). At this point, there may be bright and dark spots or part of a diaphragm obscuring the viewfield, as the illumination configuration is still not complete.

Now that the basic components of the microscope are set, the instrument is ready to be configured for Khler illumination. The first step is to narrow the size of the field diaphragm and translate the condenser up and down via the adjustment knob until you see a sharp image of the edges from field diaphragm leaves (Figure 5(a)). If this strategy doesn't work, change the size of the field diaphragm

and try again. In many cases, the condenser is not yet centered so only one edge of the viewfield will contain elements of the field diaphragm leaves.

At this stage, the field diaphragm is in sharp focus, but the condenser is not yet centered. The centering knobs on the microscope condenser mounting frame are used to center the condenser (Figure 5(b)). Close down the field diaphragm to its smallest opening and use the centering screws to relocate the bright opening to the center of the viewfield (a crosshair reticule is very useful for determining the center of the viewfield). Once the condenser is centered, open the field diaphragm until its leaves move completely out of the viewfield.

At this point in the procedure, you should almost have a suitable image in the viewfield. The only remaining task is to optimize the contrast. Remember that you first opened the aperture diaphragm of the condenser in order to see more light (discussed above). The aperture size must now be corrected in order to improve contrast and to seek a suitable balance between contrast and resolution. Remember that closing the condenser aperture too far will seriously degrade resolution and significantly darken the image.

You can directly visualize the aperture diaphragm superimposed over the objective rear focal plane (objective pupil) if you remove an eyepiece from the binocular tube mount and peer directly into the tube (Figure 6(a)). Your eye should be between 10 and 20 centimeters away from the tube for the best observation. While looking down the tube, open and close the condenser aperture diaphragm until you can clearly recognize the image in the objective pupil. Finally, set the diameter of the diaphragm in such a manner that it illuminates between 65 and 80 percent of the pupil diameter (see Figure 6(b)), providing nearly full resolution and optimum contrast. Such a compromise is usually made in choosing the resolution because it is primarily contrast that renders the image acceptable to the eye. After the condenser aperture diaphragm adjusted, insert the eyepiece back into the binocular observation tube mount.

The microscope should now be properly configured for observation of specimens in Khler illumination, but only for the objective that was used to set the instrument. When another objective is rotated into the optical train, you should always adjust the condenser aperture and field diaphragm for optimum illumination (in effect, contrast versus resolution) conditions. However, it is usually not necessary to remove the eyepiece each time a new objective is used. Once the aperture diaphragm size has been set for the 10x objective, it is often sufficient to adapt the size for other objectives by visualizing the image. First open the aperture until the stop is reached, and then close it down slowly until the image begins to get slightly darker and the contrast simultaneously increases. Often, the most important parameter is to achieve suitable contrast. From the above discussion it is evident that the condenser aperture diaphragm should be set to a position that will provide a compromise mixture of direct and deviated light that depends, to a large degree, on the absorption, diffraction, and refraction characteristics of the specimen. This must be accomplished without overwhelming the image with artifacts that obscure detail and present erroneous enhancement of contrast. The amount of image detail and contrast necessary to produce the best photomicrograph is also dependent upon refractive index, optical characteristics, and other specimen-dependent parameters. When the aperture diaphragm is erroneously closed too far, deviated light begins to obscure direct illuminating rays, resulting in diffraction artifacts that cause visible fringes, banding, and/or pattern formation in photomicrographs. Other problems, such as refraction phenomena, can also produce apparent structures in an image that are not real. Alternatively, opening the condenser aperture too wide causes unwanted glare and light scattering from the specimen and optical surfaces within the microscope. This leads to a significant loss of contrast and washing out of image detail. The correct setting will vary from specimen to specimen, and the experienced microscopist will soon learn to accurately adjust the condenser aperture diaphragm (and numerical aperture of the system) by observing the image without having to view the diaphragm in the back focal plane of the objective. In fact, many microscopists believe that critical reduction of the numerical aperture of the microscope system to optimize image quality is the single most important step in photomicrography. The illumination system of the microscope, when adjusted for proper Khler illumination, must satisfy several requirements. The illuminated area of the specimen plane must be at least as large as the field of view for any given objective. Also, the light must be of uniform intensity and the numerical aperture must vary from a maximum (equal to that of the objective) to a minimum value that will depend upon the optical characteristics of the specimen.