Appendix E: Image Analysis

Figure E.1. Schematic of image analysis system

Figure E.2. Capabilities of Olympus CUE 2 system

Computers and video recorders have been combined in a powerful combination for advanced histochemical work. A video camera can be attached to a microscope and coupled to a frame- grabber board within a personal computer. The image can then be captured and displayed on a video monitor in either black & white, color, or pseudocolor. The latter is an enhancement feature of the system which allows assignment of a color to a chosen gray scale of a black & white image. That is, the color is not real, but an assigned value, determined by the operator or the computer system. Since the eye can detect more colors than tones of gray, it increases the ability to visually distinguish subtle tone differences.

A sample system suitable for the undergraduate laboratory is represented by the CUE 2 system manufactured by Olympus. The system consists of:

Image analysis systems used to be too expensive for routine work, but the price is steadily decreasing. A generic system can actually be pieced together from commercially available components for under $10,000. A fully integrated package will cost slightly more, and a real time system (one which captures and analyzes the image as it is generated) can increase the price by a factor of 10. You get a lot of power for the investment, however.

Image analysis begins with a digital capturing of the image as data. In an intermediate system suitable for cell work, the image is detected with 256 gray levels (the human eye can detect only 9) and displayed with a resolution of either 256 x 256 or 512 x 512 pixels. A pixel is an abbreviation for picture elements, and represents the dots on a screen, or more significantly, the number of information points in the image. A 512 x 512 pixel image thus contains 262,144 pieces of information. Each piece of information will in turn be stored as 8, 16 or 32 bits in computer memory, depending on the computer system attached. An 8 bit machine (IBM PC or equivalent) will satisfy the low end of this scale, a 16 bit (IBM AT or 286 clone) the intermediate, while the upper end will require a significantly faster computer (386 based, or minicomputer). For an undergraduate laboratory (and most research work), the information stored in the low end system (256 x 256, 8 bit) is sufficient, with the high end reserved for analysis of true color and/or real time images. A display resolution of 512 x 512 pixels makes the image sharper appearing and easier to work with.

Once the image is acquired in digital form, it becomes data which can be manipulated for changes in the display image, or for statistical purposes. The operational capabilities of the CUE 2 system, as listed in Figure E-2, demonstrate many of these features.

Software options for image analysis include packages for densitometry (replacing microspectrophotometers), 3-D construction of serial sections, production of stereo images, autoradiographic analysis, planar morphometry including linear analysis (line length, width and angles) in addition to the features listed for the basic system.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 --