Optical sectioning with a fluorescence confocal SLM: procedures for determination of the 2‐D digital modulation transfer function and for 3‐D reconstruction by tessellation

We have determined the operational parameters of a confocal scanning laser microscope (CSLM) used for fluorescence imaging. The system performance was characterized by the modulation transfer function (MTF), calculated from an edge response function (ERF) corresponding to a standard test pattern overlaying fluorescence solutions on a microscopic slide. Signal truncation error was avoided by making the ERF continuous at its limits through superposition of a suitable linear curve. We determined an appropriate scanning step density by defining a compromise between the requirements of the sampling theorem with respect to aliasing and the need for minimal suppression of higher spatial frequencies. These procedures for choosing the sampling density of the total digital CSLM permit a systematic optimization of the image acquisition parameters.

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