Spectral Modeling in Fluorescence Microscopy
Neil Anderson, Ph.D., Prashant Prabhat, Ph.D., Turan Erdogan, Ph.D.
Fluorescence microscopy is a ubiquitous and continuously evolving technique which permits one to peer into the biological world at the micron length scale and beyond, allowing direct access into the spatial location and behavior of small molecules at the cellular level. This level of performance can be achieved only through careful instrument design and the use of highquality and high-performance optical components, such as optical filters. Optical filters transmit and block (via reflection) light over specific, well-defined spectral ranges. Filters with poor transmission, edge steepness, and blocking offer limited performance and, in the case of fluorescence microscopy, can result in the acquisition of images with poor contrast, thus limiting the ability to reveal the secrets of the subcellular world. To avoid this limitation, microscopists have been rapidly switching to modern hard-coated thin-film interference filters. Given the critical role optical filters play in fluorescence microscopy, it is important to understand how such filters transmit both the desired fluorescence signal as well as the undesired background light (i.e., optical “noise”). Two critical performance criteria for any optical system that generates, captures, and records fluorescence light are the level of desired light signal detected and the level of...
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