Color Photography

The most frequent problem encountered in color photomicrography is matching the colors recorded on film to those perceived by the observer through the eyepiece. In particular the color of the photographic image will depend on the color temperature of the light source. Tungsten filaments give off a reddish light, which will not be obvious to the observer, but will be clear in photomicrographs taken with a standard "daylight"

An object stained:

Filter used

Blue

Green

Yellow

Red

becomes:

Red

Very dark

Dark

Light

Very light

Green

Dark

Very light

Light

Very dark

Blue

Very light

Dark

Dark

Very dark

Cyan

Light

Light

Dark

Very dark

Magenta

Light

Very dark

Light

Light

Yellow

Very dark

Light

Very light

Light

film. Films designed for tungsten light sources (e.g., Fujichrome 64T) are therefore adjusted for a color temperature of or 3200 K. Some microscope lamps have a "PHOTO" or "3200 K" setting, which produces a standardized bright illumination for photography. Manufacturers may alternatively supply a color temperature vs voltage curve, which enables lamp intensity to be adjusted accordingly. The high color temperatures required for color constancy (usually equivalent to a lamp burning at its brightest) may demand impossibly rapid shutter speeds. Since altering the brightness of the lamp would also change the color temperature of the illumination, light intensity can only be reduced by placing neutral density filters in the light path. These absorb energy equally across the spectrum and can be used additively to achieve a reasonable photographic exposure time without changing color temperature.

Daylight films are adjusted for a color temperature of 5500-6000 K and when used with tungsten illumination, a blue filter (Kodak Wratten 80A) can compensate for the orange cast of artificial lighting conditions (Fig. 4A). Daylight films should be used for color fluorescence photomicrographs (UV lamps have a higher color temperature), although, unless two different fluorochromes are being photographed, black-and-white film generally gives better resolution and exposure reliability than color film (see Subheading 3.9.1.). Typical films are listed below. All are color-reversal films (producing transparencies).

FUJI 64 T (for standard tungsten illumination) EKTACHROME 400 ASA (fluorescence) KODAK P1600X (fluorescence)

Fig. 4. (opposite page) (A) Color temperature: The effect of a blue filter when a daylight film is used to photograph a specimen under tungsten illumination. The filter compensates for the pinkness of the illumination and increases the contrast. (B) Pseudocolor: Three examples of the use of pseudocolor with black-and-white digital images. (Top) A pseudocolor LUT is used to convert the gray scales within a "sharpened" confocal microscope image of a Dil-labeled embryonic brain into changes in color complements. (Middle) Black-and-white

1) Colour temperature

2) Pseudocolour colour look-up tables

"Sift colour look-up tables

Input Intensity

Raw image

Input Intensity combining different fluorescence wavelengths

Raw image combining different fluorescence wavelengths

488 nm

combining different optical sections

Ventricular

Sharpened

Pseudocolour

Sharpened

Pseudocolour

Combined

Fig. 4. (continued) images of the same specimen illuminated for different fluorescent dyes are color-coded and merged to produce an image equivalent to a photographic double exposure. This image is then merged with a DIC picture of the same specimen. (Bottom) Confocal microscope optical sections are color-coded and merged to show the differences in cell dispersal at different depths in the tissue. Pseudocolor pictures were prepared within Adobe Photoshop from 8-bit 768 x 512 pixel images produced on a Bio-Rad MRC-600 confocal system mounted on a Nikon Diaphot inverted compound microscope. (See color plate 4 appearing after p. 368.)

3.9.1. Reciprocity Failure

When exposure times exceed much more than maybe 2 s, the sensitivity of color-reversal films begins to change. Effectively the film's speed decreases with time, a phenomenon known as failure of reciprocity (the relationship between exposure and sensitivity). This can be corrected for by multiplying exposure times by the appropriate factor and most photomicroscopes have a built-in facility to correct for reciprocity failure. By entering the appropriate reciprocity index for a given film, exposure times are automatically adjusted for long exposures. If necessary, reciprocity failure can be determined by trial and error for a given film type (increasing exposure 2x on successive exposures). Between 2 and 8 s, there is also a shift in the sensitivity of the different color pigments within the emulsion. For example, as the green pigment becomes less sensitive, the film will take on a purplish tinge. Film manufacturers may supply details of appropriate color compensation filters within this exposure range, which can be used to correct shifts in color sensitivity. Beyond 8 s of exposure, it is unlikely that any color filters can compensate entirely for the shifts in color balance (2).

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