1 Principles of Confocal Scanning
1.6 Dynamic Range & PMT Gain and Offset
Image dynamic range is the ratio the brightest structure of interest to the dimmest. With the LeicaTCS-NT confocal microscope the dynamic range of the image is limited by the 256 levels of the 8-bit analogue to digital converter (A2D), which takes the signal from the PMT and converts it into a number. The possible range of such numbers is 1 to 256 (actually 0 to 255). In comparison the dark adapted eye has a dynamic range in excess of 1 to 10000.
Because of the limited dynamic range of the 8 bit analogue to digital converter it is crucial to set the minimum and maximum levels of the image to match the fixed range of the A2D converter closely. Good matching is achieved by carefully setting the offset and gain of the PMT so that its output makes full use of the fixed range of the A2D. A methodical approach is to use the glowunder/glowover look up table scheme, which uses blue to indicate A2D overload and green to indicate underload. First set the background to be just green using the PMT offset control, then set then PMT gain (voltage) to just produce blue pixels on the bright part of the image that is of interest. Once the offset is adjusted you will find that the value will be close to zero, and that offset will not need to be adjusted for the rest of the scanning session.
It is of interest to note that gain and offset have the same effect on digital images as contrast and brightness on computer and TV monitors.
Last updated 10/24/06
|© 1998-2003 Michael Chua, Cell & Molecular Physiology, UNC. All rights reserved.|
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