Specimen Contrast in Optical Microscopy

High resolution optical microscopy of living organisms and similar transparent, unstained specimens typically suffers from a lack of contrast, rendering these specimens nearly invisible in brightfield illumination mode. Using the full aperture of the microscope objective, images of unstained specimens are extremely poor, even for transparent periodic structures, such as diffraction gratings, aligned fibers, integrated circuit replicas, filamentous algae, and diatoms.

Although transparent specimens usually induce phase shifts to interacting light beams through scattering and diffraction, these objects remain invisible in the microscope because the human eye cannot detect differences in phase. Unless the specimen is stained with highly colored dyes, the only alternative available to the microscopist is to significantly reduce the condenser working numerical aperture by closing the iris diaphragm. However, this extreme measure will significantly impair the resolution of the objective.

The optical systems contained in modern microscopes may be capable of producing high resolution images at high magnifications, but such a capability is worthless without sufficient contrast in the image. Contrast is not an inherent property of the specimen, but is dependent upon interaction of the specimen with light and the efficiency of the optical system coupled to its ability to reliably record the image information with a suitable detector. Control of image contrast in the microscope optical system is dependent upon several factors, including proper setting of aperture diaphragms, degree of optical aberration, the contrast mechanism employed, the type of specimen, and the characteristics of the detector. Aside from specialized contrast-enhancing accessories, there are several locations in the microscope that enable the operator to adjust contrast. The most critical to the optical system are the field and condenser aperture diaphragm settings, but contrast can also be manipulated by varying electronic camera (or traditional emulsion film) gamma, altering the magnification for video detectors, processing images in real time, as well as specimen staining.

Because the human eye perceives an object by the contrast generated in its image, a certain degree of confusion can result unless there is prior knowledge of the optical events that occur to produce contrast in the image. Figure 1 illustrates a series of three digital images captured in transmitted light mode of the same viewfield containing a transparent, almost colorless Zygnema filamentous algae under differing contrast modes: brightfield, phase contrast, and differential interference contrast. The three digital images appear quite dissimilar, and because of these variations, the microscopist might arrive at a different conclusion from independent examination of each viewfield.

The algae filament illustrated in Figure 1(a) was imaged through a microscope operating in brightfield mode with the condenser aperture reduced in size enough to render the edges visible and to expose some internal detail. Although the green chloroplasts can be distinguished within the ribs of the filament, the image generally suffers from an overall lack of contrast. An identical viewfield of the filamentous algae captured with phase contrast optics is presented in Figure 1(b). Note the series of ribbed, ring-like structures that appear to be ordered in groups of two and the high-contrast spherical shapes that are revealed to be chloroplasts in Figure 1(a). In general, the image displays dark regions surrounded by halos, which are a common artifact in phase contrast microscopy. The filament illustrated in Figure 1(c) was imaged using differential interference contrast microscopy with a small degree of bias applied through a de Sénarmont compensator. Specimen shading introduced by the compensator results in one side of the image appearing dark while the other side is brighter, leading to the perception of a pseudo three-dimensional image. Each viewfield in Figure 1 provides a different specimen image and leads to slightly different interpretations, which can only be deciphered with knowledge about how the microscope created these images.

The absorption of light (either naturally or mediated through the addition of synthetic dyes) to produce colors or vary the brightness of a specimen has been the classical method of producing contrast in brightfield microscopy. The term contrast refers to the ability of an individual specimen detail to be distinguished when compared to the background or other adjacent features. In effect, contrast is defined as the difference in light intensity between the specimen image and the adjacent background relative to the overall background intensity. Specimen properties that produce changes in brightness, or color differences, arise from light absorption, reflection, spatial variation in refractive index, scattering, diffraction, birefringence, fluorescence and similar optical phenomena. In general, contrast is measured by the relationship between the highest and lowest intensity in an image, and can be described by a simple formula:

Percent Contrast = [(Ib - Is)/Ib] x 100

where I(b) is the background intensity and I(s) is the intensity of specimen features for which contrast is being investigated. From this equation, it is evident that specimen contrast refers to the relationship between the highest and lowest intensity in the image. If the specimen intensity is less (darker) than that of the background, contrast is referred to as being positive, while specimens that are lighter than the background display negative contrast. When a specimen modifies the spectral distribution (color) of light passing through, it produces color contrast. This type of contrast is also produced by interference of white light in specimens with closely spaced periodic structures.

The graph presented in Figure 2 illustrates the effect of background intensity on specimen contrast. When the background is a very dark gray value (I(b) equals 0.01; red line), a small change in image intensity generates a large change in contrast. By lightening the background to a somewhat lighter gray (I(b) equals 0.10; green line), small changes in image intensity provide a useful range of contrast. At still lighter background intensities (I(b) is greater than or equal to about 0.50; blue line), image contrast is relatively insensitive to background intensity, and large changes in image intensity produce only small increases or decreases in contrast.

In most situations, the background and image intensities are not discrete values, but vary over the entire viewfield, leading to fluctuations in contrast. As a rule of thumb, transparent specimens in brightfield illumination mode display about 2 to 5 percent contrast, while phase and differential interference contrast images can have contrast levels between 15 and 20 percent, just slightly less than that observed with fixed and stained specimens in brightfield illumination (about 25 percent). Compared to darkfield and fluorescence microscopy (average contrast levels of 60 and 75 percent, respectively), phase contrast and differential interference contrast illumination produce less overall contrast but still afford a similar degree of resolution.

Amplitude and Phase Specimens

Prior to the invention of optical contrast enhancement techniques, transmitted brightfield illumination was one of the most commonly utilized observation modes in optical microscopy, especially for fixed, stained specimens or other types of samples having high natural absorption of visible light. Collectively, specimens readily imaged with brightfield illumination are termed amplitude objects (or specimens) because the amplitude or intensity of the illuminating wavefronts is reduced when light passes through the specimen.

However, for many specimens in optical microscopy, especially unstained or living material, contrast is so poor that the specimen remains essentially invisible regardless of the ability of the objective to resolve or clearly separate details. Often, for just such specimens, it is important not to alter them by killing or treatment with chemical dyes or fixatives. This necessity has led microscopists to experiment with contrast enhancing techniques for over a hundred years in an attempt to improve specimen visibility and to bring more detail to the image without altering the specimen itself. It is a common practice to reduce the condenser aperture diaphragm below the recommended size or to lower the substage condenser in order to increase specimen contrast. Unfortunately, while these maneuvers will indeed increase contrast, they also seriously reduce resolution and sharpness.

In brightfield illumination, both the light source (usually a tungsten-halogen lamp) and condenser are positioned to fill the objective front aperture with partially coherent wavefronts that are symmetrical with respect to the microscope optical axis. Wavefronts that interact with stained regions of the specimen are reduced in amplitude, while those diffracted by the specimen produce prominent first-order diffraction side bands, which are 180 degrees out of phase with light passing through the specimen. The diffracted side bands interfere at the image plane with the surround light waves during formation of the image, and the background appears bright, with absorbing structures in the specimen exhibiting a variety of colors and gray-level tones. However, when the refractive index of an unstained specimen is similar to that of the surrounding medium (typically referred to as the surround), the intensity of diffracted light waves is dramatically reduced. In addition, the relative phase of retardation imparted to diffracted wavefronts emanating from the specimen is shifted by only 90 degrees instead of the usual 180 degrees observed with highly absorbing specimens. Both of these effects combine to severely limit visibility of the specimen.

As discussed above, specimens that alter the intensity of transmitted light are termed amplitude specimens and can be observed in the microscope as a consequence of their ability to absorb or otherwise affect the light intensity, which is proportional to the square of the light wave amplitude. Other specimens that are naturally colored or artificially stained with chemical color dyes can also be clearly imaged in brightfield illumination. These stains or natural colors absorb some portion of the white light passing through and transmit or reflect other colors. Often, stains are combined to yield contrasting colors. For example, blue hematoxylin stain for cell nuclei is often combined with pink eosin that selectively stains cytoplasm. It is a common practice to utilize stains on specimens that do not readily absorb light, thus rendering such images visible in the microscope.

In contrast, transparent specimens that do not absorb light, but instead, produce a phase change to wavefronts passing through are termed phase objects (or specimens). These specimens are virtually invisible and very difficult to image because the human eye is insensitive to changes in the relative phase shifts between visible light waves. In addition, the eye is unable to detect orientational changes, such as polarizing effects, in the electric field vectors comprising electromagnetic radiation. Phase specimens are characterized by several criteria including their shape (typically round or flat), the density of internal light scattering elements, thickness, and unique chemical or electrical structural properties (collectively grouped as refractive index). Thick specimens may be relatively transparent and contain only a few light scattering elements, or they may contain many scattering elements that do not pass light and render the specimen effectively opaque to transmitted illumination. These specimens are often termed reflected light specimens.

Phase changes are primarily due to differences in thickness and refractive index between the specimen and its surrounding medium. A typical example is living tissue culture cells, which appear almost transparent under brightfield illumination. When passing through a transparent specimen, the illuminating light wavefront is not modified in a brightfield microscope, nor is the image-forming wave absorbed by the specimen. If the specimen is somewhat out of focus, a thin grayscale shadow produced by refracted light is observed through the eyepieces. However, when the microscope is properly focused, the image of a thin, transparent specimen disappears (specimens having a significant thickness continue to be observed due to refracted light from numerous focal planes along the optical or z-axis).

The effect on incident illuminating light rays of amplitude and phase specimens is presented in Figure 3. The uppermost sine wave in the figure illustrates a typical undisturbed (surround) light wave that does not pass through the specimen. The amplitude and wavelength of the undisturbed light wave passing only through the specimen mounting medium (having refractive index = n) are indicated in the illustration (Figure 3(a)). When light waves enter a stained specimen (represented by the green box in Figure 3(b)) having the same refractive index as the surrounding medium, the waves experience a reduction in amplitude as a result of absorption by the stain, but their relative phase remains unchanged. However, when light waves enter a specimen having a refractive index that is different from the surrounding medium (Figure 3(c)), the amplitude is not affected, but the phase is retarded by approximately 90 degrees. In reality, a majority of the specimens encountered exhibit a combination of amplitude and phase effects (Figure 3(d)), producing changes to both the amplitude and phase relationships between the incident and emerging light waves.

Optical Path Differences and Phase Gradients

Optical path differences and phase gradients experienced by incident wavefronts passing through a transparent specimen are utilized to full advantage by several popular contrast enhancing techniques, including phase contrast and differential interference contrast. In most cases, the portion of an incident wavefront that traverses the specimen, but not through the surrounding medium, is either slightly advanced or retarded, depending upon the refractive index differential between the specimen and the medium. When considering transparent phase specimens that are poorly imaged in brightfield illumination, the role of the specimen in altering the optical path length (in effect, the relative phase shift) of waves passing through is of paramount importance.

A majority of phase specimens that are observed in culture vessels or sandwiched between a microscope slide and coverslip are relatively flat or plate-like as illustrated in Figure 4 for a hypothetical specimen immersed in a homogeneous medium. In this figure, the specimen has a thickness denoted by the variable t and a refractive index, n(s). The refractive index of the surrounding medium is n(m). Incident light waves (yellow arrows) approach the specimen, bathed in its surrounding medium, from the left and pass through at a velocity dictated by the product of the refractive index and thickness.

When light passes from one medium into another (for example, from an aqueous nutrient tissue culture medium into the cytoplasm of a cell), the velocity is altered according to the refractive index differences between the two media. Thus, when a coherent light wave emitted by the focused microscope filament passes through a phase specimen having a thickness t and a refractive index n(s), the wave is either increased or decreased in velocity. If the refractive index of the specimen is greater than that of the surrounding medium, the wave is reduced in velocity while passing through the specimen and is subsequently retarded in relative phase when it emerges from the specimen. Alternatively, when the refractive index of the surrounding medium exceeds that of the specimen, the wave is advanced in phase upon exiting the specimen.

Light traveling exclusively through the specimen experiences an optical path (OP) that is determined by the product of the specimen refractive index (n(s)) and the specimen thickness (t). In a similar manner, light waves traversing only through the surrounding media travel an optical path distance equal to the thickness of the medium multiplied by the refractive index (t × n(m)). The optical path difference (OPD) between the specimen and its surrounding medium can be expressed as:

Optical Path Difference (OPD) = Δ = (t × ns - t × nm) = t × (ns - nm)

The difference in location of an emergent wavefront between the specimen and surrounding medium is termed the phase shift (δ) and can be defined in radians by the following equation:

δ = 2πΔ/λ

where π is a constant (3.142) and λ is the wavelength of light illuminating the specimen. The optical path difference is the product of two terms: the thickness (t) and the difference in refractive index (n). In many cases, the optical path difference can be quite large even though the thickness of the object is quite thin. On the other hand, when the refractive indices of the specimen and the surrounding medium are equal, the optical path difference is zero even if the specimen thickness is very large. In this case, light traveling through the specimen is merely delayed (a phase difference) relative to the light passing an equal thickness of the surrounding medium.

The phase contrast microscope is designed to take advantage of phase differences between the various components in a specimen and the surrounding medium. However, it is not simply a phase difference that is necessary, but also diffraction by the specimen must occur for the phase contrast microscope to produce a suitable image. By comparison, differential interference contrast relies on phase gradients to generate contrast in otherwise transparent specimens, resulting in the classical pseudo three-dimensional images for which the technique is widely known.

The most common shape of a phase object is one of continuously changing optical path or density, such as the hemispherical specimen illustrated in Figure 5. In this example, the sides of the phase object can be approximated mathematically by a prism shape, as discussed below. The refractive index of the phase object in Figure 5 is designated n(s) and that of the surrounding medium, n(m). Radical geometrical transitions in shape for the phase object occur only at edges A and B (see Figure 5(a)). Incident light impacts the object perpendicular to the plane AB, while the plane wavefront is parallel to AB.

The boundary at 1 (the apex of the rounded phase object in Figure 5(a)) is essentially parallel to the incident wavefront whereas the boundaries at A and B are perpendicular. Regions 2 through 4 are miniature prisms defined by a tangent to the rounded surface of the phase object (Figure 5(b)). The "prism" angle is lesser at region 2 than at region 4, which is opposite to region 4'. At edges A and B diffraction is strongest.

Thus, curved or hemispherical specimens are composed of many prisms, and opposite sides of the specimen have prisms oriented in opposite directions. The steepest prisms are at the equator of a spherical specimen, while prisms with the least slope are located at the top and bottom. These miniature prisms form an optical gradient:

OG (Optical Gradient) = Φ = α(ns - nm)

Where α is the angle of the tangent with respect to the plane wavefront. Notice that the optical gradient is the product of two terms: the angle (α) between the sides that the light passes, and the difference in refractive index (n(s) - n(m)). Light that passes through a prism changes direction by the angle φ (see Figure 5(c) and 5(d)). The change in direction can be large, even though the slope or boundary gradient may be small, if the difference in refractive index is large. If the refractive indices are identical, the light wave passes through the phase object unrefracted. The direction of light exiting the prism is dependent on the relative difference in refractive indices between the phase object (n(s)) and its surrounding medium (n(m); see Figure 5(c) and 5(d)).

As discussed above, rounded phase objects have continuously varying optical gradients, and each individual optical gradient "prism" creates a different angle of light deflection. Figure 5(c) illustrates the direction of deflection when the surrounding medium has a refractive index greater than the phase object (n(m) > n(s)), and Figure 5(d) shows the direction when the opposite is true (n(m) < n(s)). The angle of deflection, φ, is proportional to the tangent angle (α) and the difference in refractive index (n(s) - n(m)), such that:

tan φ = tan α (ns - nm)

For small angles:

φ = α (ns - nm) (in radians)

To summarize the "prism" effect of optical gradient boundaries, when the refractive index of the phase object exceeds that of the surrounding medium, then gradients of equal size on each slope of the object deflect at the same angle. This deflection angle is dependent upon the relative refractive index difference and the geometric tangent angle (α). When the refractive index of the medium (n(m)) is greater than the refractive index of the object (n(s)), the deflection is opposite from when the situation is reversed. When there is no gradient, there is no deflection of light passing through.

The edges, gradients, and thickness of a specimen, regardless of whether the specimen is roughly classified as amplitude or phase, affect the refraction and diffraction angles of light wavefronts passing through. Only a portion of this scattered light is captured by the objective, a factor that is dependent upon the numerical aperture. The remaining scattered light, which is not collected, represents specimen information that is lost to the resulting image. The aperture of the objective rear focal plane mimics a low pass filter (affecting large specimen details) for diffracted light, which must be focused at the intermediate image plane to undergo interference and form an image. Because smooth rounded surfaces of phase specimens have relatively few or no diffraction sites, they suffer a significant lack of contrast when imaged without the benefit of auxiliary contrast enhancing optical components.

Coherency of Illumination

When considering optical methods to enhance specimen contrast, it is useful to consider various characteristics of a specimen that can be manipulated to create intensity variations that will result in rendering the specimen visible. A primary question is which characteristic of the object will be transformed into a difference of intensity under a unique set of circumstances.

Minute specimen details and edges that have a size approximating the wavelength of imaging light will diffract or scatter light, provided there is a difference in refractive index between the specimen and its surrounding medium. Refractive index is classically defined as the ratio of the speed of light through air or a vacuum divided by the speed of light through the object. Because the speed of light through any material is less than the speed of light in a vacuum, the refractive index always exceeds a value of 1.0 for specimens examined with a microscope. In order to resolve small distances between objects and to reproduce their shape with reasonable fidelity, a large angle of diffracted light must be captured by the microscope objective.

Diffracted (or deviated) light gathered by the objective must be brought into a sharp focus at the image plane in order to generate specimen detail. At the image plane, light waves comprising the diffracted light undergo interference with undiffracted light that passes through and around the specimen. The quality of the image generated by interference is highly dependent upon the coherency of light illuminating the specimen, and image quality generally is dramatically improved by increasing the coherence. In the optical microscope, the condenser aperture diaphragm opening size partially controls the spatial coherence of light incident on the specimen. Decreasing the diaphragm opening size yields a greater spatial coherence.

Illumination of the specimen with light waves that are at least partially coherent is critical to the role of diffraction and interference in image formation, and is required in all forms of interference optical microscopy, including phase contrast, differential interference contrast (DIC), and polarization. In effect, the light waves passing through the specimen (surround or undeviated) and those diffracted by the specimen have a mutual degree of coherent character, which must be preserved throughout the microscope optical train to enable constructive and destructive interference to occur at the image plane.

Incandescent filament lamps, such as the tungsten-halogen type commonly employed in optical microscopes, are partially coherent light sources with a significant number of wavefronts vibrating in phase from their point of origin on the filament. Because each of the photons from an ensemble generated by a hot filament vibrates slightly out of phase with one another, the actual degree of coherence within a given wavefront is only partial. In addition, the distance over which the waves exhibit strong coherence is also limited to just a few dozen wavelengths. As a result, the long-range amplitude of a light wave will exhibit alternating high and low states, where the waves vibrate coherently (in phase) and are transiently out of phase with each other, respectively. The situation with filament and arc lamps can be compared to stimulated emission from laser sources, which is coherent over long distances (many thousands or even millions of wavelengths), and completely incoherent fluorescence emission generated by fluorochrome-labeled specimens.

The origin of coherence is derived from the fact that atoms within a microscopic domain in the lamp filament strongly influence each other when they are excited to the point that photons are emitted. These short-range effects lead to localized synchronous emission that occurs in discrete bundles over the entire filament surface. As a result, the hot filament of a tungsten-halide incandescent lamp, or the ionized plasma of an arc-discharge mercury burner, can be described as an ensemble of miniature atomic neighborhoods that independently emit orchestrated bursts of light. Individual coherent wavefronts generated by the filament along a single azimuth are termed a ray or pencil of light. In Köhler illumination, the collector lens (positioned after the lamphouse, but before the field diaphragm in the microscope optical pathway) forms an image of the filament in the front focal plane (aperture) of the substage condenser. This aperture subsequently becomes the source of partially coherent wavefronts that are utilized to illuminate the specimen in the object plane, as illustrated in Figure 6.

For a given light wave that is incident on the specimen (see inset in Figure 6), a coherence relationship exists between the diffracted and undiffracted (zeroth-order) wavefronts passing through the specimen. This relationship is maintained between the specimen and the image plane, where the light waves recombine and interfere with each other. At that location, the coherent wave partners add incoherently with other wavefronts to produce the final image. This important concept underlies many forms of optical microscopy, but is especially critical to phase and differential interference contrast techniques, because image formation through constructive and destructive interference requires that the illumination be at least partially coherent. In other words, a definite phase relationship must exist between the surround and diffracted light waves, and this relationship must be preserved between the specimen and the image plane.

Microscopists have long relied on decreasing the condenser aperture diaphragm opening size to increase visibility of particles and edges in phase specimens. Contrast for amplitude objects can also be improved by proper adjustment of the condenser aperture. Small objects, edges, and particles will diffract light regardless of whether they belong to an amplitude or phase specimen. Only a portion of this diffracted light is captured by the objective (due to numerical aperture limitations), and the remaining diffracted light that is not collected represents image information that is lost. The correct setting for the condenser aperture diaphragm opening size is a tradeoff between enhancement of specimen image contrast and the introduction of diffraction artifacts. The latter are manifested in a loss of resolution, superimposition of diffraction rings, and other undesirable optical effects originating from regions in the specimen that are not in common focus. As the diffraction limit is approached, image contrast is compromised as object details become smaller and spatial frequencies become higher.

In the optical microscope, contrast is derived as a result of the interaction between illuminating light wavefronts and the specimen, and how the light leaving the specimen is processed, but is not an inherent property of a particular specimen. Furthermore, the modulation transfer function (MTF) and contrast-generating mode of the microscope significantly affects specimen contrast. The modulation transfer function is a quantitative indicator of the ability of the microscope to transfer information from the specimen to the image. In general, image contrast decreases for specimen structures that have higher spatial frequencies (finer and more closely spaced details), regardless of the microscope contrast mechanism or modulation transfer function.

Conclusions

Light can interact with a specimen through a variety of mechanisms to generate image contrast. These include reflection from the surface, absorption, refraction, polarization, fluorescence, and diffraction. Contrast can also be increased by physical modification of the microscope optical components and illumination mode, as well as manipulation of the final image through photographic or digital electronic techniques. The following discussion highlights various interactions between the specimen and light, and reviews some of the optical microscopy techniques (see Table 1) that have been developed to enhance specimen contrast. Table 1 presents a summary of the contrast enhancing technique(s) of choice for a variety of specimens and materials that are studied with both transmitted and reflected light microscopy. This table may be used as a rough guide to approach specific imaging problems in optical microscopy.

The human eye is very sensitive to amplitude and wavelength differences in a specimen. For this reason, many specimens are cut into very thin sections (ranging from 1-30 microns in thickness) and stained with chemical dyes to increase contrast and to differentiate between structures residing within the specimen. This technique has been quite commonly used with biological specimens for several hundred years. Dyes selectively absorb light from one or several wavelengths and pass or reflect all other wavelengths. An example is a blue dye that absorbs all visible light wavelengths with the exception of blue, which is reflected from and transmitted through the specimen. Internal structural elements of a biological specimen are often stained with a mixture of dyes to selectively stain these elements, increasing their contrast against a background of material that is either transparent or stained a different color.

These techniques also apply to fluorescent dyes, which can be used to increase contrast in specimens with a high degree of specificity. When fluorescent specimens are stimulated by one or several wavelengths of visible or near-ultraviolet light, they often emit longer wavelengths of light and thereby become visible or contrasted relative to other objects in the field or the dark background.

Contrast-Enhancing Techniques for Optical Microscopy
Specimen
Type
Imaging
Technique
Transmitted Light
Transparent Specimens
Phase Objects

Bacteria, Spermatozoa,
Cells in Glass Containers,
Protozoa, Mites, Fibers, etc.
Phase Contrast
Differential Interference Contrast (DIC)
Hoffman Modulation Contrast
Oblique Illumination
Light Scattering Objects
Diatoms, Fibers, Hairs,
Fresh Water Microorganisms,
Radiolarians, etc.
Rheinberg Illumination
Darkfield Illumination
Phase Contrast and DIC
Light Refracting Specimens
Colloidal Suspensions
powders and minerals
Liquids
Phase Contrast
Dispersion Staining
DIC
Amplitude Specimens
Stained Tissue
Naturally Colored Specimens
Hair and Fibers
Insects and Marine Algae
Brightfield Illumination
Fluorescent Specimens
Cells in Tissue Culture
Fluorochrome-Stained Sections
Smears and Spreads
Fluorescence Illumination
Birefringent Specimens
Mineral Thin Sections
Liquid Crystals
Melted and Recrystallized Chemicals
Hairs and Fibers
Bones and Feathers
Polarized Illumination
Reflected Light
Specular (Reflecting) Surface
Thin Films, Mirrors
Polished Metallurgical Samples
Integrated Circuits
Brightfield Illumination
Phase Contrast, DIC
Darkfield Illumination
Diffuse (Non-Reflecting) Surface
Thin and Thick Films
Rocks and Minerals
Hairs, Fibers, and Bone
Insects
Brightfield Illumination
Phase Contrast, DIC
Darkfield Illumination
Amplitude Surface Features
Dyed Fibers
Diffuse Metallic Specimens
Composite Materials
Polymers
Brightfield Illumination
Darkfield Illumination
Birefringent Specimens
Mineral Thin Sections
Hairs and Fibers
Bones and Feathers
Single Crystals
Oriented Films
Polarized Illumination
Fluorescent Specimens
Mounted Cells
Fluorochrome-Stained Sections
Smears and Spreads
Fluorescence Illumination
Table 1

An early, but still currently employed method of increasing contrast in stained specimens utilizes color contrast filters, Wratten lacquered gelatin squares (from Kodak), or interference filters in the light path. For example, if a specimen is stained with a red stain, a green filter will darken the red areas, thus increasing contrast. On the other hand, a green filter will lighten any green stained area. Color filters are very valuable aids to specimen contrast, especially when black and white photomicrography is the goal. Green filters are particularly valuable for applications with achromat objectives, which are spherically corrected for green light. In addition, the internal phase plates in phase contrast objectives are designed to retard or advance specific wavelengths, usually in the neighborhood of 550 nanometers (green light).

Living specimens and other phase objects, which often yield poor images when viewed in brightfield illumination, are made clearly visible by optical rather than chemical means when viewed under phase contrast, Hoffman modulation contrast, and differential interference contrast illumination. These techniques require special optical components in the microscope, but will generally produce images of sufficient contrast to reveal important details about specimen structure.

Another simple technique for contrast improvement involves the selection of a mounting medium with a refractive index substantially different from that of the specimen. For example, diatoms can be mounted in a variety of contrast-enhancing mediums such as air or the commercial medium Styrax. The difference in refractive indices improves the contrast of these colorless specimens and renders their outlines and markings more visible.

Anisotropic materials usually exhibit two or more refractive indices and are thus termed birefringent, or doubly refracting. Among the specimens included in this category are minerals, crystals (which exhibit a high degree of structural symmetry), fibers, hairs, and a wide spectrum of other biological specimens. When light enters a non-optic (an axis other than the optic axis) axis of anisotropic crystals, it is refracted into two rays each polarized with their vibration directions oriented at right angles to one another, and traveling at different velocities. The resulting light waves are polarized and can interfere when recombined in the microscope analyzer to produce images of birefringent specimens that are highly colored and contain a significant amount of contrast.

When incident illumination strikes an opaque surface, it is reflected in a manner that is specific to the terrain of that surface. Very smooth surfaces reflect light at an angle equaling that of the incident light, a mechanism known as specular reflection. Uneven or diffuse surfaces tend to reflect light at all possible angles by a phenomenon known as diffuse reflection, resulting in a reduced amount of light entering the objective. Contrast in reflected light microscopy can be enhanced by careful specimen preparation. Metallographic samples are often etched with reactive liquids and gases to reveal grain boundaries and/or polished to increase the amount of light reflected into the microscope. Stains, in the form of fluorescent dyes, thin films, and metallic coatings, are also used to introduce contrast in reflected light microscopy specimens. Birefringent specimens can be imaged using polarized reflected light and transparent phase objects are often the subject of observation using techniques such as reflected differential interference contrast, darkfield illumination, and Hoffman modulation contrast.

Regardless of the microscope technique employed to generate specimen contrast, the three fundamental parameters that govern image quality are resolution, magnification, and contrast. The primary function of the microscope is to increase the size and resolve minute specimen details, but this act must be accomplished with sufficient contrast to render the specimen visible to the detector (a digital or traditional camera or the human eye).


BACK TO PHASE CONTRAST MICROSCOPY

Contributing Authors

Douglas B. Murphy - Department of Cell Biology and Anatomy and Microscope Facility, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, 107 WBSB, Baltimore, Maryland 21205.

Robert Hoffman - Modulation Optics, Inc., 100 Forest Drive, Greenvale, New York 11548.

Kenneth R. Spring - Scientific Consultant, Lusby, Maryland, 20657.

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