Quantitative Fluorescence Microscopy: Considerations and Controls

It is important to have a working awareness of the many factors that can enhance, degrade or even distort the interpretation of quantitative data. Measurements in fluorescence microscopy may be discussed in the context of three major headings: (1) intensity, (2) spatial, and (3) temporal. The quantitative ability of instrumentation in each dimension is dependent on the performance characteristics of the instrument subsystems that contribute to the data gathering. In order for accurate and precise data to be recorded, not only must each subsystem perform well on its own merits, they must all be carefully orchestrated to work together in synergy. A number of basic considerations regarding the quantitative application of imaging instruments is outlined in the pages that follow; topics covered include detector technologies, illumination sources, optical limits, scan raster, specimen positioning and multi-channel acquisition.

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