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Fluorescence Microscopy Contrast

[#10] Last update 4 Feb 19

Overview of Contrast in Fluorescence Microscopy

Pete Kepf, Certified Vision Professional- Advanced


Machine vision feature extraction for fluorescence microscopy faces the same challenges as other applications. However, because of the low lumen levels involved and the sensitivity required, signal-to-noise (S/N) is a chief concern. S/N is affected by the illumination, the optics and the dynamic range of the camera.

Text Box: Figure 2: Contrast (Courtesy Chroma Technology Corporation)We have seen that illumination consists of emitted light in the form of fluorescence. Because the intent is to isolate a specific wavelength to determine the material properties of a specimen, the less range of wavelength values seen by the detector, the better the results will be. Therefore, contrast is more desirable than simply brightness. Figure 2 illustrates two levels of contrast. Both filters pass the desired wavelength, but the one on the right also passes much more undesired light. This is seen as noise. Better analytical results will be obtained from the left-hand image due to higher S/N.

Because of their degree of magnification, microscopy applications have notoriously shallow depths of field. If you took high school or college biology, you’ll recall having to frequently adjust the microscope lens up and down from the slide to maintain focus. The same laws of optics apply to scientific instruments.