When describing digital images, gray-level resolution is a term that refers to the number of shades of gray utilized in preparing the image for display. Digital images having higher gray-level resolution are composed with a larger number of gray shades and have a greater dynamic range than those of lower gray-level resolution.

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The tutorial initializes with a randomly selected specimen image, captured in the MIC-D digital microscope, appearing in the left-hand window entitled Specimen Image. Each specimen name includes, in parentheses, an abbreviation designating the contrast mechanism employed in obtaining the image. The following nomenclature is used: (BF), brightfield; (DF), darkfield; (OB), oblique illumination; and (RL), reflected light. Visitors will note that specimens captured using the various techniques available with the MIC-D microscope behave differently during image processing in the tutorial.

Adjacent to the Specimen Image window is a Current Resolution window that displays the captured image at various gray-level resolutions that are adjustable with the Grayscale Bit Depth slider. To operate the tutorial, select an image from the Choose A Specimen pull-down menu, and vary the gray-level resolution with the Grayscale Bit Depth slider. The number of bits utilized in the displayed image is presented directly above the slider, as is the total number of gray levels.

The Olympus MIC-D digital microscope is equipped with a CMOS image sensor that captures images having 640 x 480 pixel dimensions in red, green, and blue (RGB) true color mode with 8-bit resolution per channel, resulting in a total of 256 tonal levels for each of the three colors. When the color images are converted to grayscale by an image-editing software program, they produce corresponding black and white (monochrome) images that have a single 8-bit channel with 256 gray levels.

The gray-level resolution of a digital image is related to the gray-level intensity of the optical image and the accuracy of the digitizing device used to capture the optical image. The gray-level intensity of an optical image refers to the amount of light energy actually reflected from or transmitted through the physical specimen. The range of gray levels utilized in creating a digital image is known as the dynamic range of the image, and is a function of both the gray-level intensity of the optical image as magnified by the microscope and the accuracy of the camera system used to capture the image.

The optical image is captured by the camera system in a process called sampling. The number of pixels contained in a digital image is governed by the distance between pixels, termed the sampling interval, which is a function of the accuracy of the digitizing device. The numerical value of each pixel in the digital image represents the intensity of the optical image averaged over the sampling interval. In the sampling process, the gray-level intensity of each sample of the optical image is measured as an analog signal by the camera. Following the sampling process, each image sample is transformed from an analog signal into a digital brightness value by a device known as an Analog to Digital converter (or A/D converter). The accuracy of each digital value is dependent in part upon the number of bits available to the converter, which determines the number of shades of gray that will be used to represent the brightness levels of the image.

In the tutorial, as the Grayscale Bit Depth slider is moved to the left, the number of bits used to display the image in the Current Resolution window decreases. The result is that the image begins to take on a mechanical appearance with significantly less detail. This phenomenon is termed gray-level contouring or posterization. Gray-level contouring becomes apparent in the background regions first, where gray levels tend to vary more gradually, and is indicative of inadequate gray-level resolution. For a majority of the typical applications in optical microscopy, 6- or 7-bit resolution is usually adequate for a visually pleasing digital image. The tutorial allows a maximum display of 7 bits because on most computers, a Web browser limits the displayed grayscale range to 5 or 6 bits, regardless of the computer screen resolution. Therefore, effects occurring in digital images at higher resolutions will not be apparent in the tutorial. However, for many quantitative applications in microscopy (such as high-resolution fluorescence), bit depths of 10, 12, or even higher (1024, 4096, or more gray levels), are necessary to capture and display all of the important specimen details. For this reason, it is often desirable to use the highest available camera resolution when capturing images in the microscope.

Image processing algorithms, such as background subtraction, compression, and histogram manipulation, which are often performed prior to display or printing can cause a loss of bit depth in the processed image. For applications requiring image deconvolution, feature classification, measurement, or other image analysis techniques, high bit depth is essential. X-ray imaging usually requires 12-bit gray-level resolution, due to the very fine differences in brightness that exist in these images, and optical densitometry involving high densities requires at least 12 to 14 bits. Because the number of bits available to the analog-to-digital converter is typically fixed, it is important to choose a digital camera or other digitizing device with brightness resolution that is adequate for the application.

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