It is no exaggeration to say that almost the entire art and science of microscopy (if specimen preparation is not taken into account) depends on the correct use of the luminous field and aperture diaphragms. Thankfully, there are simple rules for this. Subsequent discussions in this section describe in detail how to correctly set the microscope for Köhler illumination. The technique is recommended by all manufactures of modern laboratory microscopes because it can produce specimen illumination that is uniformly bright and free from glare, thus allowing the user to realize the microscope's full potential. It will be much easier for you if you have made yourself familiar with the relationships between the conjugate planes. The aperture planes (also known as pupils) are responsible for resolving power and contrasting techniques and can be controlled by light filters and diaphragm settings. In contrast, the field planes contain images formed by the optical components of the microscope and are the home for reticles and scales used to measure lengths. Field planes are controlled via the field diaphragm.

Today, many microscope manufacturers offer advanced models that permit the user to alternate or simultaneously conduct investigations using both vertical and transmitted illumination. A typical microscope configured for both types of illumination is illustrated in Figure 4 (the transmitted light source and optical pathway is not shown in this illustration). The optical pathway for reflected light begins with illuminating rays originating in the lamp housing for reflected light (the upper housing in Figure 4). This light next passes through the collector lens and into the vertical illuminator where it is controlled by the aperture and field diaphragms. After passing through the vertical illuminator, the light is then reflected by a beamsplitter (a half mirror or elliptically shaped first-surface mirror) through the objective to illuminate the specimen. Light reflected from the surface of the specimen re-enters the objective and passes into the binocular head where it is directed either to the eyepieces or to a port for photomicrography. Reflected light microscopy is frequently the domain of industrial applications, especially in the rapidly growing semiconductor arena, and thus represents a most important segment of microscopical studies.

Fluorite objectives are produced from advanced glass formulations that contain materials such as fluorspar or newer synthetic substitutes. These new formulations allow for greatly improved correction of optical aberration. Similar to the achromats, the fluorite objectives are also corrected chromatically for red and blue light. In addition, the fluorites are also corrected spherically for two or three colors instead of a single color, as are achromats. The superior correction of fluorite objectives compared to achromats enables these objectives to be made with a higher numerical aperture, resulting in brighter images. Fluorite objectives also have better resolving power than achromats and provide a higher degree of contrast, making them better suited than achromats for color photomicrography in white light.

Another reason for the existence of fixed and adjustable diaphragms, prisms, beamsplitters, and filters in the microscope is that the illumination often should be reset after each change of the objective. This is, in part, because the size of the observed specimen field changes with every objective magnification. An objective with a low magnification (for example, 4x) provides a large field of observation (with a diameter as large as 5 millimeters in this case, provided that the eyepiece permits to observe an intermediate image with a diameter of 20 millimeters). If a switch is made to the 40x objective, the diameter of the viewfield of the specimen shrinks by the factor 10 (to only 0.5 millimeters). The viewable area then becomes as much as 100x smaller. The second reason is that the numerical aperture increases from 0.12 to 0.65 or, expressed as aperture angles, from 15 degrees to 80 degrees. The effect of numerical aperture on illumination light cone size and shape is presented in Figure 1.

Erin E. Wilson and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

The standard thickness for cover glasses is 0.17 millimeters, which is designated as a number 1½ cover glass. Unfortunately, not all 1½ cover glasses are manufactured to this close tolerance (they range from 0.16 to 0.19 millimeters) and many specimens have media between them and the cover glass. Compensation for cover glass thickness can be accomplished by adjusting the mechanical tube length of the microscope, or (as previously discussed) by the utilization of specialized correction collars that change the spacing between critical elements inside the objective barrel. The correction collar is utilized to adjust for these subtle differences to ensure the optimum objective performance. Proper utilization of objective lenses with correction collars demands that the microscopist is experienced and alert enough to reset the collar using appropriate image criteria. In most cases, focus may shift and the image may wander during adjustment of the correction collar. Use the steps listed below to make small incremental adjustments to an objective's correction collar while observing changes in the specimen image.

The least expensive (and most common) objectives, employed on a majority of laboratory microscopes, are the achromatic objectives. These objectives are corrected for axial chromatic aberration in two wavelengths (blue and red; about 486 and 656 nanometers, respectively), which are brought into a single common focal point. Furthermore, achromatic objectives are corrected for spherical aberration in the color green (546 nanometers; see Table 1). The limited correction of achromatic objectives can lead to substantial artifacts when specimens are examined and imaged with color microscopy and photomicrography. If focus is chosen in the green region of the spectrum, images will have a reddish-magenta halo (often termed residual color). Achromatic objectives yield their best results with light passed through a green filter (often an interference filter) and using black and white film when these objectives are employed for photomicrography. The lack of correction for flatness of field further hampers achromat objectives. In the past few years, most manufacturers have begun providing flat-field corrections for achromat objectives and have given these corrected objectives the name of plan achromats.

The field diaphragm in the base of the microscope controls only the width of the bundle of light rays reaching the condenser. This variable aperture does not affect the optical resolution, numerical aperture, or the intensity of illumination. Proper adjustment of the field diaphragm (in effect, centered in the optical path and opened so as to lie just outside of the field of view) is important for preventing glare than can reduce contrast in the observed image. The elimination of excess light is particularly important when attempting to image samples with inherently low contrast. When the field diaphragm is opened too far, scattered light originating from the specimen and light reflected at oblique angles from optical surfaces can act to degrade image quality.

Modern objectives, made up of many glass elements, have reached a high state of quality and performance, with the extent of correction for aberrations and flatness of field determining the usefulness and cost of an objective. Construction techniques and materials used to manufacture objectives have greatly improved over the course of the past 100 years. Today, objectives are designed with the assistance of Computer-Aided-Design (CAD) systems using advanced rare-element glass formulations of uniform composition and quality having highly specific refractive indices. The enhanced performance that is demonstrated using these advanced techniques has allowed manufacturers to produce objectives that are very low in dispersion and corrected for most of the common optical artifacts such as coma, astigmatism, geometrical distortion, field curvature, spherical and chromatic aberration. Not only are microscope objectives now corrected for more aberrations over wider fields, but image flare has been dramatically reduced with a substantial increase in light transmission, yielding images that are remarkably bright, sharp, and crisp.

To remedy this, many high-performance apochromat dry objectives are fitted with correction collars, which allow adjustment to correct for spherical aberration by correcting for variations in cover glass thickness (see Figure 5). Optical correction for spherical aberration is produced by rotating the collar, which causes two of the lens element groups in the objective to move either closer together or farther apart. The objective on the left in Figure 5 has had the correction collar adjusted for a cover glass thickness of 0.20 mm by bringing the adjustable lens elements very close together. In contrast, the objective on the right in Figure 5 has the adjustable lens elements separated by a rather large distance to compensate for very thin cover glasses (0.13 mm). A majority of the correction collar objectives designed for upright transmitted light microscopy have an adjustment range for cover glass thickness variations between 0.10 and 0.23 millimeters. Many of the specialized phase contrast objectives designed for observing tissue culture specimens with an inverted microscope have an even broader compensation range of 0 to 2 millimeters. This allows specimens to be viewed through the bottom of most culture vessels, which often have dramatic thickness fluctuations in this size range. Uncovered specimens, such as blood smears, can also be observed with correction collar objectives when the adjustment is set to 0 to account for the lack of a cover glass.

The microscope contains two different groups of interlaced optical planes that are responsible for controlling illumination and image formation. Collectively, these optical planes are known as conjugate planes. The first group of planes (termed the aperture planes) controls the beam path for illuminating light and produces a focused image of the lamp filament at the plane of the substage condenser aperture diaphragm, the rear focal plane of the objective, and the eye point (also called the Ramsden disk) of the eyepiece. Conjugate planes are in common focus and are critical in achieving proper Köhler illumination. A second set of planes, known as the image-forming conjugate planes include the field diaphragm, the specimen, the fixed diaphragm of the eyepiece and the retina of the eye or the surface of a camera detector. By definition, an object that is in focus at one plane is also in focus at the other conjugate planes of that light path. In each light pathway (both image-forming and illumination), there are four separate planes that together make up the conjugate plane set.

Inverted microscope stands incorporate the vertical illuminator within the body of the microscope. Many types of objectives can be used with inverted reflected light microscopes, and all modes of reflected light illumination may be possible: brightfield, darkfield, polarized light, differential interference contrast, and fluorescence. Some of the instruments include a magnification changer for zooming in on the image, contrast filters, and a variety of reticles. Because an inverted microscope is a favorite instrument for metallographers, it is often referred to as a metallograph. Manufacturers are largely migrating to using infinity-corrected optics in reflected light microscopes, but there are still thousands of fixed tube length microscopes in use with objectives corrected for a tube length between 160 and 210 millimeters.

Typesof objectivelenses

Major microscope manufacturers offer a wide range of objective designs, which feature excellent optical characteristics under a wide spectrum of illumination conditions and provide various degrees of correction for the primary optical aberrations. The objective illustrated in Figure 1 is a 60x oil immersion apochromat, which contains 15 optical elements that are cemented together into three groups of lens doublets, a lens triplet group, and three individual internal single-element lenses. The objective also has a hemispherical front lens and a meniscus second lens, which work synchronously to assist in capturing light rays at high numerical aperture with a minimum of spherical aberration. As is the case with most oil immersion objectives, the apochromat illustrated in Figure 1 is equipped with a spring-loaded retractable nosecone assembly that protects the front lens elements and the specimen from collision damage. Internal lens elements are carefully oriented and tightly packed into a tubular brass housing that is encapsulated by the objective barrel. Specific objective parameters such as numerical aperture, magnification, optical tube length, degree of aberration correction, and other important characteristics are imprinted or engraved on the external portion of the barrel. Although the objective featured in Figure 1 is designed to operate utilizing oil as the imaging medium between the objective front lens and specimen, other objectives have front lens elements that allow them to be use either in air or immersed in water, glycerin, or a other specialized hydrocarbon-based oils.

World-class Nikon objectives, including renowned CFI60 infinity optics, deliver brilliant images of breathtaking sharpness and clarity, from ultra-low to the highest magnifications.

Older objectives generally have lower numerical apertures, and are subject to an aberration termed chromatic difference of magnification that requires correction by the use of specially designed compensating oculars or eyepieces. This type of correction was prevalent during the reign of fixed tube length microscopes, but is not necessary with modern infinity-corrected objectives and microscopes. In recent years, modern microscope objectives have their correction for chromatic difference of magnification either built into the objectives themselves (Olympus and Nikon) or corrected in the tube lens (Leica and Zeiss).

Conjugate focal planes are often useful in troubleshooting a microscope for contaminating dust, fibers, and imperfections in the optical elements. If these artifacts are in sharp focus, it follows that they must reside on or near a surface that is part of the imaging-forming set of conjugate planes. Members of this set include the glass element at the microscope light port, the specimen, the reticle in the eyepiece, and the bottom lens element of the eyepiece. Alternatively, if these contaminants are fuzzy and out of focus, look for them near the illuminating set of elements that share conjugate planes. Suspects in this category are the condenser top lens (where dust and dirt often accumulate), the exposed eyepiece lens element (contaminants from eyelashes), and the objective front lens (usually fingerprint smudges).

Whatarethe3objectivelenses ona microscope

The general design of a practical oil immersion objective includes a hemispherical front lens element, followed by a positive meniscus lens and a doublet lens group. Presented in Figure 6 are the aplanatic refractions that occur at the first two lens elements in a typical apochromatic oil immersion objective. The specimen is sandwiched between the microscope slide and cover glass at point P, the aplanatic point of the hemispherical lens element. Light rays refracted at the rear of the hemispherical lens appear to proceed from point P(1), which is also the center of curvature for the first surface of the meniscus lens. The refracted light rays enter the meniscus lens along the radius of its first surface and experience no refraction at that surface. At the rear surface of the meniscus lens, light rays are refracted aplanatically, so they appear to diverge from point P(2). Refraction of the light rays at the surfaces of subsequent lens groups in the objective complete the convergence of light rays originating from point P, thus forming the intermediate image.

Objectives that use water and/or glycerin as an imaging medium are also available for applications with living cells in culture or sections of tissue immersed in physiological saline solution. Plan apochromat water immersion lenses are equipped with correction collars and numerical apertures up to 1.2, slightly less than their oil immersion counterparts. These objectives allow microscopists to focus through up to 200 microns of aqueous media and still retain excellent optical correction. The downside is that high numerical aperture water immersion lenses often cost many thousands of dollars and the image can still degrade when the objective is focused deeply through refractile tissue or cell parts. For more details on water, glycerin, and oil immersion objectives, visit the Molecular Expressions Microscopy Primer.

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, critical photomicrography, and digital imaging. Any lack of brightness is not a problem in simple brightfield microscopy, but if contrast-enhancing techniques, such as phase contrast, differential interference contrast, fluorescence, or polarization contrast are used, additional optical elements that consume a significant portion of the available light flow are inserted into the beam path. Such a situation leaves little light for observation, and as a result, the images assume a dark character. When properly adjusted, light from the condenser will fill the rear focal plane of the objective with image-forming light by projecting a cone of light to illuminate the field of view. The condenser aperture diaphragm is responsible for controlling the angle of the illuminating light cone and, consequently, the numerical aperture of the condenser. This concept is illustrated in Figure 1, where a series of condensers are illustrated with light cones (and numerical apertures) of decreasing size from left to right in the figure.

There are a wide variety of light sources available to illuminate microscopes, both for routine observation and for quantitative digital imaging. A most common light source, because of its low cost and long life, is the 30 to 100 watt tungsten-halogen lamp. These lamps are relatively bright with a color spectrum centered at 3200 Kelvin (when set close to the nominal voltage of the lamp), but require color conversion filters to raise their color temperature to daylight equivalence. Another popular light source is the 75 to 150 watt xenon arc discharge lamp because of its very high brightness and long life. Xenon lamps feature a relatively even output of intensity across the visual spectrum, and a color temperature that approximates daylight. When very high light intensity is required, metal-halide and mercury lamps, as well as lasers are often used. In fluorescence microscopy, particularly for the purpose of digital imaging, 100 watt or 200 watt mercury burners have been used for the past several decades. These lamps are slowly yielding to the more stable and longer lived metal-halide lamps, which feature higher intensity in the continuum regions. Increasingly, light emitting diodes (LEDs) are used as a source for microscope illumination in transmitted light as well as fluorescence.

What is the objective lens of a microscopegive

The imaging medium between the objective front lens and the specimen coverslip is also very important with respect to correction for spherical aberration and coma in the design of lens elements for objectives. Lower power objectives have relatively low numerical apertures and are designed to be used dry with only air as the imaging medium between the objective front lens and the cover glass. The maximum theoretical numerical aperture obtainable with air is 1.0, however in practice it is virtually impossible to produce a dry objective with a numerical aperture above 0.95. The effect of cover glass thickness variation is negligible for dry objectives having numerical apertures less than 0.4, but such deviation becomes significant at numerical apertures exceeding 0.65, where fluctuations as small as 0.01 millimeter can introduce spherical aberration. This poses problems with high-power apochromats, which must use very short working distances in air and contain sensitive corrections for spherical aberration that tend to make it difficult to obtain sharp images.

Uncorrected field curvature is the most severe optical aberration that occurs in fluorite (semi-apochromat) and apochromat objectives, and it was tolerated as an unavoidable artifact for many years. During routine use, the viewfield would have to be continuously refocused between the center and the edges to capture all specimen details. The introduction of flat-field (plan) correction to objectives perfected their use for photomicrography and video microscopy, and today these corrections are standard in both general use and high-performance objectives. Correction for field curvature adds a considerable number of lens elements to the objective as illustrated in Figure 4 with a simple achromat. The uncorrected achromat on the left in Figure 4 contains two lens doublets, in addition to a simple thin-lens front element. In contrast, the corrected plan achromat on the right in Figure 4 contains three lens doublets, a central lens triplet group, and a meniscus lens positioned behind the hemispherical front lens. Plan correction, in this instance, has led to the addition of six lens elements bundled into more sophisticated lens groupings, which dramatically increases the optical complexity of the objective. The significant increase in lens elements for plan correction also occurs with fluorite and apochromat objectives, frequently resulting in an extremely tight fit of lens elements (see Figure 1) within the internal objective sleeve. In general, plan objectives corrected for field curvature sacrifice a considerable amount of free working distance, and many of the high-magnification versions have a concave front lens, which can be extremely difficult to clean and maintain.

A typical upright compound reflected light microscope has two eyepiece viewing tubes (Figure 4) and often a trinocular tube head for mounting a conventional or digital/video camera system (not illustrated). Standard equipment eyepieces are usually of 10x magnification, and most microscopes are equipped with a nosepiece capable of holding four to six objectives. The stage is mechanically controlled with a specimen holder that can be translated in the x- and y- directions and the entire stage unit is capable of precise up and down movement with a coarse and fine focusing mechanism. Built-in light sources range from 20 and 100 watt tungsten-halogen bulbs to higher energy mercury vapor or xenon lamps that are used in fluorescence microscopy. Light passes from the lamphouse through a vertical illuminator interposed above the nosepiece but below the underside of the viewing tube head. The specimen's top surface is upright (usually without a coverslip) on the stage facing the objective, which has been rotated into the microscope's optical axis. The vertical illuminator is horizontally oriented at a 90-degree angle to the optical axis of the microscope and parallel to the table top, with the lamp housing attached to the back of the illuminator. The coarse and fine adjustment knobs raise or lower the stage in large or small increments to bring the specimen into sharp focus.

What is the objective lens of a microscopeexplain

Investigate how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for fluctuations in coverslip thickness.

Objective lensfunction

High numerical aperture dry objectives lacking a correction collar often produce images that are inferior to those of lower numerical aperture objectives where cover glass thickness is of less concern. For this reason, it is often prudent to choose a lower magnification (and numerical aperture) objective in order to obtain superior contrast without the accompanying artifacts introduced by cover glass fluctuations. As an example, a 40x objective having a numerical aperture of 0.65 may be able to produce better images with sharper contrast and clarity than a 60x-0.85 numerical aperture objective, even though the resolving power of the higher magnification objective is theoretically greater.

The substage condenser is typically mounted directly beneath the microscope stage in a bracket that can be raised or lowered independently of the stage by rotating a knurled knob. The aperture diaphragm is opened and closed with either a swinging arm, a lever, or by rotating a collar on the condenser housing. It should be noted that correct adjustment of the substage condenser is probably the most critical aspect of achieving proper Köhler illumination. Unfortunately, however, condenser misalignment and improperly adjusted condenser aperture diaphragms are the main source of image degradation and poor quality photomicrography.

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. Objectives are also instrumental in determining the magnification of a particular specimen and the resolution under which fine specimen detail can be observed in the microscope.

Microscopeparts

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 microns. The range of specimens falling into this category is enormous and includes most metals, ores, ceramics, many polymers, semiconductors (unprocessed silicon, wafers, and integrated circuits), slag, coal, plastics, paint, paper, wood, leather, glass inclusions, and a wide variety of specialized materials. Because light is unable to pass through these specimens, it must be directed onto the surface and eventually returned to the microscope objective by either specular or diffused reflection. As mentioned above, such illumination is most often referred to as episcopic illumination, epi-illumination, or vertical illumination (essentially originating from above), in contrast to diascopic (transmitted) illumination that passes through a specimen.

The advantages of oil immersion objectives are severely compromised if the wrong immersion fluid is utilized. Microscope manufacturers produce objectives with tight tolerances to refractive index and dispersion, which require matching values in the liquid placed between the cover glass and objective front lens. It is advisable to employ only the oil intended by the objective manufacturer, and to not mix immersion oils between manufacturers to avoid unpleasant artifacts such as crystallization or phase separation.

Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.

Objective lensmagnification

In most biological and petrographic applications, a cover glass is utilized in mounting the specimen, both to protect the integrity of the specimen and to provide a clear window for observation. The cover glass acts to converge the light cones originating from each point in the specimen, but also introduces chromatic and spherical aberration (and consequent loss of contrast) that must be corrected by the objective. The degree to which light rays are converged is determined by the refractive index, dispersion, and thickness of the cover glass. Although the refractive index should be relatively constant within a batch of cover glasses, the thickness can vary between 0.13 and 0.22 millimeters. Another concern is the aqueous solvent or excess mounting medium that lies between the specimen and cover glass in wet or thickly mounted preparations. For example, in physiological saline whose refractive index is significantly different from that of the coverslip, the objective must focus through a layer of water only a few microns thick, leading to significant aberrations and a deviation of the point spread function that is no longer symmetrical above and below the focal plane. These factors add to the effective variations in refractive index and thickness of the coverslip and are very difficult for the microscopist to control.

Properly designed oil immersion objective lenses also correct for chromatic defects that are introduced by the first two lens elements, while introducing a minimum amount of spherical aberration. The fact that the light cone is partially converged before entering the first lens element aids in the control of spherical aberration. It should be noted that employing an oil immersion objective without the application oil between the coverslip and first lens element results in defective images. This due to refraction that occurs at the surface of the front lens, which introduces spherical aberration that cannot be corrected by subsequent lens components within the objective.

Objective numerical aperture can be dramatically increased by designing the objective to be used with an immersion medium, such as oil, glycerin, or water. By using an immersion medium with a refractive index similar to that of the glass coverslip, image degradation due to thickness variations of the cover glass are practically eliminated whereby rays of wide obliquity no longer undergo refraction and are more readily grasped by the objective. Typical immersion oils have a refractive index of 1.51 and a dispersion similar to that of glass coverslips. Light rays passing through the specimen encounter a homogeneous medium between the coverslip and immersion oil and are not refracted as they enter the lens, but only as they leave its upper surface. It follows that if the specimen is placed at the aplanatic point of the first objective lens, imaging by this portion of the lens system is totally free of spherical aberration.

In the vertical illuminator, light travels from the light source, usually a 12 volt 50 or 100 watt tungsten-halogen lamp, passes through collector lenses, through the variable aperture iris diaphragm opening and through the opening of a variable and centerable pre-focused field iris diaphragm. The light then strikes a partially silvered plane glass reflector, or strikes a fully silvered periphery of a mirror with elliptical opening for darkfield illumination. The plane glass reflector is partially silvered on the glass side facing the light source and anti-reflection coated on the glass side facing the observation tube in brightfield reflected illumination. Light is thus deflected downward into the objective. The mirrors are tilted at an angle of 45 degrees to the path of the light travelling along the vertical illuminator.

The highest level of correction (and expense) is found in apochromatic objectives, illustrated in Figures 2 and 3. Apochromats represent the most highly corrected microscope lenses currently available, and their high price reflects the sophisticated design and careful assembly required in their manufacture. In Figure 3, we compare lens elements in a series of apochromatic objectives ranging from 10x to 100x in magnification. The lower power apochromat objectives (10x and 20x) have a longer working distance and the overall objective length is shorter than in higher power (40x and 100x) apochromat objectives. 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 (see Table 1). Apochromatic 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 numerical apertures than do achromats or fluorites. Many of the newer high-performance fluorite and apochromat objectives are corrected for four (dark blue, blue, green, and red) or more colors chromatically and four colors spherically.

Efficient sample illumination is very dependent upon proper alignment of all the optical components in the microscope, including the illumination source. The serious microscopist should become familiar with the adjustment range of each component and should practice aligning these with different samples and objectives. Uneven illumination can have a serious impact on the quality of photomicrographs and digital images, causing a variety of other undesirable effects. Some of the newest microscopes are equipped with pre-centered lamps that do not allow for adjustment, and some even supply condensers that do not have lateral adjustment mechanisms. It is important to ensure that these microscopes were aligned for proper Köhler illumination at the factory before engaging in photomicrography. Carefully read the microscope instruction manual and/or question your factory technical representative for important details about how these microscopes are optimized for illumination.

The objective is the most difficult component of an optical microscope to design and assemble, and is the first element that light encounters as it proceeds from the specimen to the image plane. Objectives derive their name from the fact that they are, by proximity, the closest component to the object (specimen) being imaged.

The next higher level of correction and cost is found in objectives called fluorites or semi-apochromats (illustrated by center objective in Figure 2), named for the mineral fluorite, which was originally used in their construction. Figure 2 depicts the three major classes of objectives: The achromats with the least amount of correction, as discussed above; the fluorites (or semi-apochromats) that have additional spherical corrections; and, the apochromats that are the most highly corrected objectives available. The objective positioned on the far left in Figure 2 is a 10x achromat, which contains two internal lens doublets and a front lens element. Illustrated in the center of Figure 2 is a 10x fluorite objective having several lens groups including two doublets and a triplet, in addition to a hemispherical front lens and a secondary meniscus lens. On the right in Figure 2 is a 10x apochromat objective that also contains multiple lens groups and single elements. Although similar in construction to fluorite objectives, the lenses have different thicknesses and curvatures and are arranged in a configuration that is unique to apochromat objectives.

In reflected light microscopy, absorption and diffraction of the incident light rays by the specimen often lead to readily discernible variations in the image, from black through various shades of gray, or color if the specimen is colored. Such specimens are known as amplitude specimens and may not require special contrast methods or treatment to make their details visible. Other specimens show so little difference in intensity and/or color that their feature details are extremely difficult to discern and distinguish in brightfield reflected light microscopy. The latter specimens behave much like the phase specimens so familiar in transmitted light work, and are suited for darkfield and reflected light differential interference contrast applications.

Objective lens microscopefunction

All three types of objectives suffer from pronounced field curvature and project images that are curved rather than flat, an artifact that increases in severity with higher magnification. To overcome this inherent condition arising from curved lens surfaces, optical designers have produced flat-field corrected objectives, which yield images that are in common focus throughout the viewfield. Objectives that have flat-field correction and low distortion are called plan achromats, plan fluorites, or plan apochromats, depending upon their degree of residual aberration. Such correction, although expensive, is quite valuable in digital imaging and conventional photomicrography.

The optical train and numerous other components of a modern microscope are presented in a cut-away diagram in Figure 2. The condenser (containing the aperture diaphragm) and the luminous-field diaphragm normally contained in the stand base are the critical elements in achieving Köhler illumination. Image forming light rays passed through the specimen are captured by the microscope objective and directed either into the eyepieces and/or to one of the several camera ports. Throughout the optical train of the microscope, illumination is directed and focused through a series of diaphragms and lenses as it travels from the light source to illuminate the specimen and then into the eyepieces or camera attachment. 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 itself is not focused at the level of the specimen, illumination at the specimen level is essentially grainless and extended (in effect, producing a uniform field of illumination), and does not suffer deterioration from dust and imperfections on the glass surfaces of the condenser. The setting of the condenser's aperture diaphragm, along with the numerical aperture of the objective, determine the realized numerical aperture of the entire microscope system. The luminous-field diaphragm determines which portion and size of the specimen is illuminated. The aperture diaphragm of the condenser is imaged on the pupil of the objective and regulates the illumination of this pupil. The entire optical system is designed in such a manner that aperture angles of the light cones are correctly set together with the aperture diaphragm.

The intermediate image in an infinity-corrected system appears at the reference focal length (formerly, the optical tube length) behind the tube lens in the optical pathway. This length varies between 160 and 250 millimeters, depending upon design constraints imposed by the manufacturer. The magnification of an infinity-corrected objective is calculated by dividing the reference focal length by the focal length of the objective lens.

Many years ago, carbon arc lights or zirconium bulbs were used to achieve high levels of illumination, but these antique sources are seldom seen today because the lamps reduced the quality and homogeny of light reaching the sample. Köhler illumination defeats many of the limitations encountered with incandescent and arc discharge light sources by creating parallel beams that pass through the specimen in an organized manner. The Köhler strategy requires that nothing more than the viewed field of the specimen is illuminated, since the excessive light outside the field of view contains scattered light that reduces contrast. At the same time, however, one of the most critical aspects of microscopy is that the light cone of the illumination should always be matched to the angular aperture of the objective to allow the entire aperture of the optics to be effectively utilized. This is the only way to achieve maximum resolving power.