Manipulation of the transfer function, and its corresponding look-up table (LUT), provides a flexible and powerful approach to adjusting the appearance of a digital image. Contrast and color values can be altered without modifying the original digitized image, and an adjustable curve may be utilized to interactively alter values present in the look-up table.

This interactive tutorial explores how manipulation of the look-up table can be employed to alter various properties of a digital image, such as contrast and color values. The tutorial initializes with a randomly selected specimen image (captured in the 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: (FL), fluorescence; (BF), brightfield; (DF), darkfield; (PC), phase contrast; (DIC), differential interference contrast (Nomarski); (HMC), Hoffman modulation contrast; and (POL), polarized light. Visitors will note that specimens captured using the various techniques available in optical microscopy behave differently during image processing in the tutorial.

Adjacent to the Specimen Image window is a Transfer Function Graph that displays a plot of the Input Pixel Brightness values versus the final Displayed Pixel Brightness values. To operate the tutorial, select a specimen image from the Choose A Specimen pull-down menu, and choose a channel of the specimen image to modify using the Choose A Channel pull-down menu. In order to display a grayscale form of the specimen image, use the mouse to place a checkmark in the Grayscale Image checkbox positioned in the lower left corner of the tutorial window.

When the Fitted Curve checkbox option is selected, translatable point nodes may be inserted into any location on the Transfer Function Graph window by clicking on the desired location adjacent to the curve with the mouse cursor. The added nodal points, including the endpoints, may be moved by dragging and dropping them anywhere within the grid area of the window. Points may be removed from the curve by dragging them outside of the grid. When the Free-Hand Curve checkbox is selected, a free-hand curve may be drawn directly onto the Transfer Function Graph window by clicking and dragging the mouse cursor inside of the window. The free-hand curve can be smoothed by clicking on the Smooth button located under the Free-Hand Curve checkbox. The Smooth button can be clicked repeatedly to apply progressively more smoothing to the curve. If application of the smoothing function is continued, the resulting curve will eventually revert to a straight line.

In either the fitted curve or free-hand curve-drawing mode, the transfer function curve will be updated while the mouse is being dragged, but the Specimen Image window will be updated only after the mouse button is released. The Output Pixel Brightness and Input Pixel Brightness text fields may be utilized to measure coordinates on the graph by moving the mouse to any point inside of the graph window. When a nodal point is selected, by clicking it with the mouse cursor, the coordinates of the nodal point are displayed using bold characters in the input and output text fields, and override values that were previously entered using the keyboard. While a nodal point is selected, the text fields may be edited to translate the nodal point value to an exact location on the graph. After editing either text field, the keyboard Enter key must be used to move the nodal point to the new position. After any operation that selects a nodal point and causes the input and output pixel values to appear in bold characters, the cursor can be returned to coordinate read-out mode by clicking the mouse cursor inside the graph window, some distance away from the curve. Visitors should explore modifying each transfer function curve and observe the resulting effects on the appearance of the specimen image.

In the tutorial, there are five channels available for modification: RGB (grayscale), Red, Green, Blue, and Intensity (using the HSI color model). The curve corresponding to each of these separate channels represents a transfer function, which relates stored (input) brightness values present in the original digital image to displayed (output) brightness values utilizing a look-up table. The look-up table is composed of a memory storage array whose indices and entries are input brightness values and output brightness values (specified by the transfer function), respectively. Pixel brightness values for each channel of the image are reassigned by the tutorial according to the transfer function graph using the look-up table, and the updated image is then displayed in the Specimen Image window.

Manipulating the transfer function, and its corresponding look-up table, is a fast and efficient method for globally adjusting brightness, color balance, and contrast in a digital image. Another common application of look-up tables is to assign pixel brightness values to arbitrarily produce pseudocolor displays that emphasize certain image details. The most familiar application of pseudocolor is in the display of weather maps and radar images in broadcast video programs. Portions of the digitized image are divided by grayscale range and passed through separate red, green, and blue look-up tables to produce the desired display. The assignment of a particular color to a specific gray level, or grayscale range, in the image is arbitrary, and color calibration scales should be displayed near the image when quantitative information is assigned to color values. This technique is often applied in fluorescence microscopy, where combining pseudocolor encoded fluorescence images with transmitted light images can help to reveal the location of the fluorescent regions. Prior to the widespread application of digital image processing techniques, investigators were forced to capture multiple-exposure photomicrographs of fluorescence and transmitted light images or (worse) to make a single exposure containing a composite of simultaneously illuminated specimens.

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