One of the primary and favorite techniques used in all forms of optical microscopy for the past three centuries, brightfield illumination relies upon changes in light absorption, refractive index, or color for generating contrast. As light passes through the specimen, regions that alter the direction, speed, and/or spectrum of the wavefronts generate optical disparities (contrast) when the rays are gathered and focused by the objective. Resolution in a brightfield system depends on both the objective and condenser numerical apertures, and an immersion medium is often required on both sides of the specimen (for numerical aperture combinations exceeding a value of 1.0). Digital cameras provide the wide dynamic range and spatial resolution required to capture the information present in a brightfield image. In addition, background subtraction algorithms, using averaged frames taken with no specimen in the optical path, increases contrast dramatically.
Simple brightfield imaging, with the microscope properly adjusted for K鰄ler illumination (see Figure 2(a)), provides a limited degree of information about the cell outline, nuclear position, and the location of larger vesicles. However, the general lack of contrast in brightfield mode renders this technique relatively useless for serious investigations of cell structure and function. Methods that enhance contrast include differential interference contrast (DIC), polarized light, phase contrast, Hoffman modulation contrast, and darkfield microscopy (examples are illustrated in Figure 2). Several of these techniques are limited by light originating in regions removed from the focal plane when imaging thicker plant and animal tissues, while polarized light requires birefringence (usually not present to a significant degree in animal cells) to generate contrast. The adherent Chinese hamster ovary cells presented in Figure 2(a) were imaged in brightfield illumination without the assistance of optical contrast-enhancing methodology. Note the general lack of contrast when compared to other images in the figure. Individual cells are difficult to distinguish and most of the internal features (for example, the nuclei) are not visible in this image.
Differential interference contrast microscopy (Figure 2(b) and Figure 3(a)) requires plane-polarized light and additional light-shearing (Nomarski or Wollaston) prisms to exaggerate minute differences in specimen thickness gradients and refractive index. Lipid bilayers, for example, produce excellent contrast in DIC because of the difference in refractive index between aqueous and lipid phases of the cell. In addition, cell boundaries in relatively flat adherent mammalian and plant cells, including the plasma membrane, nucleus, vacuoles, mitochondria, and stress fibers, which usually generate significant gradients, are readily imaged with DIC. In plant tissues, the birefringent cell wall reduces contrast in DIC to a limited degree, but a properly aligned system should permit visualization of nuclear and vacuolar membranes, mitochondria, chloroplasts, and condensed chromosomes in epidermal cells. Differential interference contrast is an important technique for imaging thick plant and animal tissues because, in addition to the increased contrast, DIC exhibits decreased depth of focus at wide apertures, creating a thin optical section of the thick specimen. This effect is also advantageous for imaging adherent cells to minimize blur arising from floating debris in the culture medium.
Polarized light microscopy (Figure 2(f) and Figure 3(b)) is conducted by viewing the specimen between crossed polarizing elements. Assemblies within the cell having birefringent properties, such as the plant cell wall, starch granules, and the mitotic spindle, as well as muscle tissue, rotate the plane of light polarization, appearing bright on a dark background. The rabbit muscle tissue illustrated in Figure 2(f) is an example of polarized light microscopy applied to living tissue observation. Note that this technique is limited by the rare occurrence of birefringence in living cells and tissues, and has yet to be fully explored. As mentioned above, differential interference contrast operates by placing a matched pair of opposing Nomarski prisms between crossed polarizers, so that any microscope equipped for DIC observation can also be employed to examine specimens in plane-polarized light simply by removing the prisms from the optical pathway.
The widely popular phase contrast technique (as illustrated in Figure 2(c) and Figure 4(a)) employs an optical mechanism to translate minute variations in phase into corresponding changes in amplitude, which can be visualized as differences in image contrast. The microscope must be equipped with a specialized condenser containing a series of annuli matched to a set of objectives containing phase rings in the rear focal plane (phase contrast objectives can also be used with fluorescence, but with a slight reduction in transmission). Phase contrast is an excellent method to increase contrast when viewing or imaging living cells in culture, but typically results in excessive halos surrounding the outlines of edge features. These halos are optical artifacts that often reduce the visibility of boundary details. The technique is not useful for thick specimens (such as plant and animal tissue sections) because shifts in phase occur in regions removed from the focal plane that distort image detail. Furthermore, floating debris and other out-of-focus phase objects interfere with imaging adherent cells on coverslips.
Often metaphorically referred to as "poor man's DIC", Hoffman modulation contrast is an oblique illumination technique that enhances contrast in living cells and tissues by detection of optical phase gradients (see Figure 2(d) and Figure 4(b)). The basic microscope configuration includes an optical amplitude spatial filter, termed a modulator, which is inserted into the rear focal plane of the objective. Light intensity passing through the modulator varies above and below an average value, which by definition, is then said to be modulated. Coupled to the objective modulator is an off-axis slit aperture that is placed in the condenser front focal plane to direct oblique illumination towards the specimen. Unlike the phase plate in phase contrast microscopy, the Hoffman modulator is designed not to alter the phase of light passing through; rather it influences the principal zeroth order maxima to produce contrast. Hoffman modulation contrast is not hampered by the use of birefringent materials (such as plastic Petri dishes) in the optical pathway, so the technique is more useful for examining specimens in containers constructed with polymeric materials. On the downside, HMC produces a number of optical artifacts that render the technique somewhat less useful than phase contrast or DIC for live-cell imaging on glass coverslips.
The methodology surrounding darkfield microscopy, although widely applied for imaging transparent specimens throughout the nineteenth and twentieth centuries, is limited in use to physically isolated cells and organisms (as presented in Figure 2(e)). In this technique, the condenser directs a cone of light onto the specimen at high azimuths so that first-order wavefronts do not directly enter the objective front lens element. Light passing through the specimen is diffracted, reflected, and/or refracted by optical discontinuities (such as the cell membrane, nucleus, and internal organelles) enabling these faint rays to enter the objective. The specimen can then be visualized as a bright object on an otherwise black background. Unfortunately, light scattered by objects removed from the focal plane also contribute to the image, thus reducing contrast and obscuring specimen detail. This artifact is compounded by the fact that dust and debris in the imaging chamber also contribute significantly to the resulting image. Furthermore, thin adherent cells often suffer from very faint signal, whereas thick plant and animal tissues redirect too much light into the objective path, reducing the effectiveness of the technique.
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