Spectral - Black And White 16x
In black and white photography through the microscope, filters are used primarily to control contrast in the final image captured on film. Specimens that are highly differentiated with respect to colored elements from biological stains are translated into shades of gray on black and white film and will often appear to have equal brightness. When this occurs, important specimen details may be lost through a lack of contrast. Filtration techniques for black and white film are significantly different from those employed in color photomicrography.
Spectral - Black and White 16x
To better image specimens using black and white photomicrography, filters are usually employed to selectively absorb one or several colors, especially when using panchromatic films that have equal sensitivity to more than one stain color. Many stained biological specimens exhibit very pale colors against a bright background (using brightfield transmitted light microscopy), which will appear as light gray tones on a white background when recorded on black and white film. To enhance contrast, a color filter is added to the microscope light path that absorbs the stained specimen color, rendering it a darker gray. Contrast can be adjusted in this manner by selectively choosing filters that absorb varying amounts of the stain color. This concept is explored with the photomicrographs presented in Figure 1, which illustrate a thin section of Solanum tuberosum (potato) stained with a quadruple stain mixture containing Safranin O (stains nuclei, chromosomes, lignified and cutinized cell walls red), Fast Green (stains cytoplasm and cellulose cell walls green), Crystal Violet (stains starch grains purple), and Orange G (stains acidophilic cytoplasm and cell walls yellow to green).
The photomicrograph illustrated in Figure 1(a) was taken with brightfield illumination and neutral density filters using Fujichrome 64T color transparency (reversal) film. This image shows starch grains in the tuber matrix stained predominantly with Safranin O and Crystal Violet, while the remainder of the specimen consists of cellulose and cell walls stained green with Fast Green and Orange G. Figure 1(b) shows the same field photographed using Kodak T-Max 100 (a continuous-tone panchromatic black-and-white negative film), but with a Kodak Wratten number 25 red filter (see Table 2) inserted into the microscope light path. Red areas of the specimen are reduced in contrast by the filter, which also introduces too much contrast in specimen details that are stained blue and green.
As a general rule when employing color filters in black and white photomicrography, utilize filters that are complimentary to specimen stain color (they absorb most of the predominant wavelengths transmitted by the stain) to maximize the amount of contrast in final images. To achieve a medium level of contrast, use filters that only partially absorb colors displayed by features of interest. Finally, to reduce contrast to a minimum, use filters that have colors identical to those of the specimen. A combination of filters can be used to enhance detail contrast in specimens stained with more than one color.
Most biological specimens lack sufficient color and contrast to be readily imaged in the optical microscope using brightfield illumination. These specimens do not usually absorb visible light to any great extent (they are not good amplitude specimens) and can be seen as a rough outline with some internal detail only when the condenser aperture size is reduced, often to the point of introducing optical artifacts. To circumvent this problem, microscopists often treat biological cells and tissues with reactive organic dyes that will selectively stain and color various portions of the biological architecture. Because the background often appears white or very light gray in brightfield microscopy, the stained tissue will appear colored and superimposed over a light background. This is often sufficient to render the details of interest visible and with enough contrast to provide good photomicrographs.
A compilation of common biological stains along with their visible light absorption properties is presented in Table 1. Dyes used by microscopists to stain biological tissue have a wide spectrum of colors, ranging from deep blue to bright red, with a myriad of intermediate colors also available. After examining the table, it will become evident that many dyes have similar spectral properties and, at first glance, it would seem that similar dyes can be used interchangeably to stain biological specimens. In many situations, similar dyes can be substituted without adverse affects, but in some cases substitute dyes can interact with other dyes or biological structures in unforeseen ways to yield less than desirable results. This is common in complex stain mixtures where the chemical and physical properties of dyes must be finely tuned and matched to each other. Often, substitution of one dye for another dramatically changes the staining properties of the mixture.
As mentioned above, many dyes have similar colors and spectral properties. However, upon close examination of their visible absorption spectra, dyes that appear similar often vary markedly in their light absorption profiles. As an example, we can compare three red dyes that appear to have identical or nearly identical spectral characteristics: Darrow Red, Safranin O, and Sudan IV. Visible light absorption spectra for the three dyes are illustrated in Figure 3. Although all three dyes absorb strongly in the 430 to 580 nanometer (blue-green) region and transmit red wavelengths, their principal absorption maxima differ by as much as 40 nanometers. The spectral widths and distributions of wavelengths absorbed also differ throughout the three spectra. Darrow red strongly absorbs wavelengths in the 400 to 460 nanometer range, but Safranin O has little absorption in this region. The absorption spectrum of Safranin O is also shifted 20 nanometers towards longer wavelengths in the 580-600 nanometer region.
Optimization of image contrast in black and white photomicrography requires careful tuning of the visible light passing through the specimen and on to the film emulsion. In order to provide a suitable visual difference between elements of the specimen and the background, a working knowledge of the visible light spectral characteristics of both dyes and color filters is essential. The absorption spectra of common biological stains are published in a variety of sources, but two of the best works are the Sigma-Aldrich Handbook of Stains, Dyes, and Indicators and H. J. Conn's Biological Stains. Table 1 contains a listing of the most commonly used biological stains and the spectral range of useful visible light transmittance for each dye.
Color filters are available from professional photographic suppliers and microscope manufacturers in the form of glass mounts or gelatin squares. The most popular series of filters for black and white photomicrography are the Kodak Wratten gelatin filters, which are indexed with a standard numbering system (see Table 2). These filters are ideal for critical photomicrography with a large number of organic dyes available, each of which can be accurately standardized with the amount of dye present per unit area. Filters made by other manufacturers can be referenced to the Kodak numbering system to establish equivalent filtration properties.
When filters are added to the light path, film exposure time must be adjusted (usually increased) to compensate for light absorption by the filter. Each filter has a designated Filter Factor that is used as a rough guide to exposure adjustment when using that filter (see Table 2). A variety of conditions work together to influence the effect on exposure by a given filter, including the spectral sensitivity of the film, light source color temperature, and the visible absorption spectra of the specimen and stain.
Because automatic camera systems will compensate for the increased density when filters are placed into the light path, a filter factor exposure addition is usually not required. However, in many instances subtle differences in spectral sensitivities of the photomultiplier or photo diodes that measure incoming light intensity to the camera can lead to incorrect exposures. Deeply colored filters may slightly displace the microscope focus, due to optical characteristics of the objectives, so carefully check focus after adding filters to the light path. Always visually inspect the final photomicrographs and manually perform any necessary adjustments.
Interference filters can also be utilized to enhance contrast in black and white photomicrography. These filters are fabricated using several evaporated thin layers of metallic alloys on optical-grade glass, and are highly accurate with respect to spectral transmission and absorption characteristics. Interference filters are designated by both the center and width of the wavelength band that is transmitted. They are commercially available from optics supply distributors or from the microscope manufacturers.
For the purpose of choosing filters and stains for black and white photomicrography, the visible light spectrum can be divided into three primary regions, which lie in the wavelength range between 400 and 700 nanometers. Wavelengths between 400 and 500 nanometers appear visually as blue light when viewed together. Likewise, wavelengths between 500 and 600 nanometers appear green, and those between 600 and 700 nanometers appear red. A dye that absorbs all wavelengths between 400 and 500 nanometers will pass only longer wavelengths between 500 and 700 nanometers (green and red light), and will appear yellow, which is the primary subtractive color obtained by mixing equal portions of green and red light. If a dye transmits light between 400 and 500 nanometers and absorbs light of longer wavelengths (500 to 700 nanometers), then it will appear blue. 041b061a72