The Digital Sight camera control unit (CCU) provides a number of image processing functions that enable the microscopist to quickly and easily correct imaging problems that arise from low or high contrast, poor sharpness, insufficient or uneven illumination, specimen shading or discoloration, and noise.

Shading, which is defined as the variation in the amplitude of the output signal current when a video sensor is uniformly illuminated, is commonly manifested as an output image that appears brighter or darker on one side with a continuously varying shade across the field, or the field may vary in brightness from the center outward toward the periphery. Shading may result from nonuniform detector sensitivity, although the problem is often caused or exacerbated by uneven specimen illumination. Shading correction or compensation is typically performed by increasing the signal amplitude or detector sensitivity in order to counteract the undesired brightness variation.

The Digital Sight camera system is equipped with a shading correction function that can be used to compensate for insufficient illumination around the periphery of the image. Included in the software are five levels of compensation ranging from a relative value of 10 percent to 50 percent in steps of 10 percent, which correct for a reduction in luminous intensity from the central part toward the screen periphery. The Digital Sight system also provides a method to correct for nonsymmetrical unevenness of illumination, which can be saved in either of two separate memory locations. The saved corrections can be recalled later for application to other images. A typical example of shading correction is illustrated in Figure 1. The shading correction feature can be accessed through the CAM Advn. menu.

Random image noise is a commonly encountered problem, especially in fluorescence microscopy, due to the low light levels that often accompany this increasingly popular technique. The presence of random noise can seriously degrade the spatial resolution and overall quality of a digital image. One method of reducing or eliminating noise is to average image frames in order to increase signal-to-noise ratio, at a small sacrifice in temporal resolution. The Digital Sight camera system includes a noise reduction feature that can be accessed through the CAM Advn. menu. Because image averaging tends to leave an afterimage on the image monitor, noise reduction should be turned off when imaging a moving subject or if faster response is needed for focusing. Figure 2 illustrates the effects of image averaging in reducing noise in digital images captured using the Digital Sight camera system.

Gamma processing or correction, referred to as tone selection, is a function that is useful for improving image brightness and contrast, especially for digital images that display a reduced dynamic range. In order to allow tone adjustment that is appropriate for various types of microscopic imaging techniques and a wide variety of specimen types, the Digital Sight camera system is equipped with seven different tone settings. When changing the tone settings, the effect should be confirmed on the display monitor in order to select the most suitable one for the specimen being imaged. The tone settings and possible applications for each are described briefly as follows:

• Tone 1 - Wide Dynamic Range: This tone setting is suitable when gradual, even tonal gradations are required, and detail in highlight areas and reflections is important. Specimens having a full scale of tonal gradations from dark to light, such as integrated circuit chips, mechanical parts and component-mounted circuit boards are appropriate for this setting.

• Tone 2 - Weak Contrast: This setting is suitable for controlling excessive contrast in imaging natural specimens under uniform illumination when the microscope is properly configured. In situations where the substage condenser aperture diaphragm is closed excessively, the Weak Contrast setting can often compensate by reducing image contrast. However, inherent focus problems may still arise.

• Tone 3 - Standard Contrast: With this setting, the image contrast is increased, resulting in more distinct image features with many specimens. This is the default setting and should be utilized when the microscope is adjusted for Köhler illumination and the substage condenser aperture diaphragm is opened to achieve the correct numerical aperture.

• Tone 4 - Strong Contrast: When this setting is selected, the contrast is increased even more than with the Standard setting. This is an appropriate choice to enhance contrast when the condenser aperture is wide open or when specimens lacking in contrast (transparent) are imaged under conditions that inherently produce low contrast, such as brightfield illumination.

• Tone 5 - Linear: No gamma compensation is applied with this setting, and the output is linearly proportional to the camera input. This algorithm is appropriate when further image processing is intended, or when quantitative comparisons of intensity data are being made.

• Tone 6 - Metallic Specimen Applications: This tone setting is provided for examination of metallic or ceramic specimens in order to suppress specular highlights while maintaining overall high contrast with clearly distinguished bright and dark areas.

• Tone 7 - Enhanced Contrast: This setting is utilized to enhance contrast, and is especially suitable for imaging materials or specimens with inherently low contrast. For example, transparent specimens, such as living cells and tissues imaged in brightfield illumination will benefit from this contrast gain. The setting may be beneficial in applications requiring recognition or counting of features that are made more distinct when imaged with maximum contrast.

In some cases, it may be desirable to apply a special effect to a digital image in order to increase the visibility of certain specimen details, or to provide a more effective presentation. To facilitate this type of modification, the Digital Sight camera control unit is capable of displaying the output image either in the normal color mode or after modification by any of four other color effects settings. A brief description of the available effects follows (refer to the figures below for details about each setting).

• Color: In this mode, the image is displayed in the natural color scheme that is observed in the microscope eyepieces (Figure 3). For the majority of images captured with the Digital Sight system, the normal color output is the most effective mode for accurate and effective reproduction of all specimen details.

  

• Nega: The Nega (an abbreviation for the common photographic term Negative) effect displays a brightness- and color-inverted form of the image, where red, green, and blue values are converted into their complementary colors (Figure 4). The technique is useful with specimens for which color inversion can be of benefit in exposing subtle details, or in quantitative analysis of specimens.

  

• Blue Back: This mode represents the black portions of the Nega image in blue (Figure 5), and is often useful to reveal details in specimens having a high degree of contrast. As a special effect, the Blue Back mode can be beneficial as a presentation tool.

  

• B & W: An abbreviation for the term Black & White, this mode displays a grayscale form of the image (Figure 6). It can be effectively used for monochromatic images such as those acquired with differential interference contrast or phase contrast techniques. In many cases, digital images destined for publication in scientific journals must first be converted into black & white renditions of those captured in full color. The B & W filter can often aid the microscopist in preparing images for publication or oral presentation.

  

• Sepia: This effect is essentially a monochrome image version displayed in sepia (brownish) tones instead of grayscale (Figure 7). The Sepia mode is more likely to be utilized in general photographic applications than in microscopy, although the effect may enhance the visibility of specimen detail in certain instances.

Various adjustments to color balance are often effective for improving digital image quality. Color adjustments can be implemented through different approaches, but the basic mechanism is to increase or decrease the relative levels of red, green, and blue in the digital image, and to vary the color intensity. The Digital Sight camera control unit provides, through the CAM Advn. menu, adjustment buttons for color density (intensity) and hue. The color density is variable through a range of -50 to +50, with positive values corresponding to increased density and negative values corresponding to decreased density. The hue control is utilized to make adjustments to the spectral components of the colors present in the image, and allows making minute adjustments to hue on an arbitrary scale ranging from -50 to +50. Figure 8 presents an example of an image that was corrected by using color-balancing functions.

A slightly blurred or out-of-focus image can often be improved by applying some form of digital processing for sharpness enhancement. Six sharpness levels are provided by the Digital Sight camera system software, ranging in relative value from 0 (off) through 5 (strong) in increasing order of the sharpening effect applied to the digital image. The increase in apparent sharpness is achieved through a contour enhancement algorithm, and may be effective in improving feature recognition even in properly focused images, although the possibility of introducing artifacts to the image should be carefully monitored. Figure 9 provides an example of sharpening a blurred or out-of-focus image.

The shading, noise reduction, tone, effects, color balance, and sharpness controls can be accessed from the CAM menu, and further information on their operation may be found in the following sections:

• CAM Menu Features - This important menu contains a number of features useful for measuring and calibrating image focus, controlling exposure mode and time, and adjusting camera sensitivity.

• Room 1 Network Client Interface - Providing a network-based remote access to the Digital Sight's camera control unit, this virtual room allows a remote operator to download live microscope images from the camera control unit. Other features include an image annotation function and an electronic zoom function. An option is also available to allow the remote user to take limited control of the camera control unit.