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Environmental Imaging

Digital imaging in the real world with an SLR digital camera requires consistency in approach. It's most important when real colors and contrasts are meant to be exact representations of the scene and when the representations are compared over time.

Several steps need to be taken to ensure consistency and accuracy. Of immediate importance is to acquire images of the scene with the correct exposure and aperture setting so that grayscale and color values remain within the dynamic range of the camera.

Exposure Time and Aperture

Exposure refers to the total amount of light that falls on the detector when taking a photograph, and is determined by the size of the lens opening through which the light passes (aperture) and the length of time that the shutter is open (exposure time, or shutter speed). Exposure time is measured in fractions of a second. Aperture values are expressed as the ratio of the focal length of the lens to the diameter of the aperture opening. Thus, a setting of f/16 means that the diameter of the aperture will be 1/16 the focal length. Larger values result in smaller apertures and greater depth of focus.

Exposure and aperture values can be determined by using a light metering system in the camera. The method for choosing a metering pattern should be reviewed by reading the accompanying manual. A metering pattern can be chosen according to the appropriate scene or specimen.

Metering systems can call for exposures that lead to the clipping of white values. Determine if the camera tends to overexpose when metering indicates correct values by photographing scenes in which small areas of white are present. Measure the whites in Photoshop and determine if these values are clipped. If so, a feature on many professional cameras can be set to consistently underexpose by a chosen value.


Consistency and accuracy of results require conditions in which the lighting is consistent. The ideal setting is when a single camera is used under controlled conditions with a light source such as strobe or flash that is consistent over time and that can overpower other light sources. Flash units are very consistent as long as they are fired several times for stabilization before important images are acquired (Figure 4.19). Flash-on-camera units are effective when multiple objects being photographed are in the same plane: They are not effective when objects are nearer and farther away because the difference in brightness alters the lightness of colors.

Figure 4.19

Figure 4.19 Parasitic mushrooms growing on a stump photographed over time with a strobe (flash) unit. Note the consistency in color and contrast.

Most settings involve lights that fade over time or conditions in which lighting changes, such as outdoor photography. Consequent shifts in color temperature create inconsistent color interpretation of scenes.

Three methods can aid in consistent color interpretation:

  • Manual white balancing
  • Reference standards
  • Saving in the Raw format (if available)—not in the JPEG or TIFF format—and then using the Adobe Camera Raw plug-in for Photo-shop for color interpretation

Manual White Balancing for Consistent Results

Professional (and some consumer) cameras will have options to manually white balance. This is preferred over generic settings for various conditions (e.g., Outdoor, Cloudy, Incandescent, etc.) because manual white balancing provides greater accuracy at determining the color temperature of the light source. A white or gray card, or a card with increments of gray, can be purchased from a professional photography store. Alternatively, a white disk—available at professional photography stores—can be mounted in front of the lens for the same purpose. The nonreflective, flat white or gray cards (or disk) provide surfaces that are uniform, which is best for determining average reflectance. Find out how to white balance manually by referring to the camera's manual. The difference between using generic camera settings for white balancing and manual white balancing can be dramatic (Figure 4.20).

Figure 4.20

Figure 4.20 The image on the top shows color interpretation from a camera when a generic setting (in this instance, Cloudy) is used versus the image on the bottom when manual white balancing against a reference is used.

For manual white balancing to work with white and gray cards, reflect from the main light source. Hold the card so that it contains the reflection of the light source. When multiple light sources are used, hold the reference card close to the scene, aiming the card mostly toward the most important light source. Another way to use a white or gray card is to include it in the scene or with the specimen when practical. Later, in Photoshop, these images can be used for correcting color (see "Manual Match Color to Reference Method" in Chapter 7, "Color Corrections and Final Steps").

White balancing against a reference card or disk can produce consistent colors and contrasts, even when images are taken over time as long as the same light source is used. Consistency is far more difficult when light sources change; for example, when a specimen is photographed under tungsten light and then under daylight.

Reference Standards

A Macbeth color chart can be included for critical applications in which colors need to remain consistent (Figure 4.21). The 24-square card, available in credit card size to larger dimensions, is adequate for most imaging applications. It can be used for calibrating or as a reference from the first session. When used as a reference, changes to colors in subsequent sessions can be used to adjust colors and brightness to the scene or specimen in the first session.

Figure 4.21

Figure 4.21 A 24-patch Macbeth color chart is included with the scene as a reference against which to create consistent color and contrast readings.

JPEG, TIFF, and Raw

Images that are saved as JPEGs and TIFFs will be color balanced, and contrast and color corrected in camera acquisition software. While that automatic process saves time, it can lead to inconsistent results (Figure 4.22).

Figure 4.22

Figure 4.22 Two images saved as JPEG on different days after manual white balancing show different interpretations of color.

Raw files, on the other hand, contain only the original values of red, green, and blue at each sensor position. Remember that each sensor in a mosaic chip (the kind of chip used for professional consumer cameras and many scientific cameras as well) is filtered by a single red or green or blue filter. The estimate of color at each position is based on information gathered about neighboring sensor values, and then an estimate of the true color is made (called demosaicing). Allowing software to demosaic while providing user adjustments gives users not only more flexibility, but a way to record values for settings. These values can then be applied to all images.

Photoshop includes demosaicing software in the form of a plug-in called Adobe Camera Raw (ACR). This plug-in opens automatically when opening Raw files (though ACR can be used when opening TIFF or JPEG files: see "Opening Images in Adobe Camera Raw" in Chapter 6). The ACR plug-in includes settings for color and contrast corrections, and is often cited as a superior means for editing Raw images.

Though off-camera, demosaicing programs are more robust, color interpretation still occurs, and some automated functions need to be reset. ACR may interpret the color temperature of the scene incorrectly and add a tint if an overall color shift is detected. If the camera has been manually white balanced, the color temperature and tint may need to be reset. To use ACR, follow these steps:

  1. In the main window of ACR, make sure the Basic tab is active. From the White Balance menu, choose As Shot.
  2. Using the Tint slider, adjust so that the tint is set to zero (0).

Images taken on different days under the same lighting source, when following these steps, can be compared for similarity or differences in color (Figure 4.23). Color accuracy in longitudinal studies can be ensured when including an external reference with the specimen.

Figure 4.23

Figure 4.23 Images of the same scene taken on two separate days in outdoor conditions.

Calibrating Cameras

The advantage of ACR is in its capability to work with a number of RAW formats from various manufacturers and to calibrate each to a standard interpretation of color and brightness levels. In so doing, workgroups with several different cameras can each produce similar interpretation of colors and contrasts.

To create a calibration, first white balance manually. Then photograph a Macbeth color chart in daylight, tungsten, or flash (strobe) lighting, depending on typical conditions for scenes or specimens. The colors and brightness level values are subsequently matched from the image of the Macbeth chart to known values. The known values are taken from the gamut of colors used to view images on computer screens, which is mentioned in more detail at

This calibration can be saved and then applied to images in ACR.

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