Physics of Pixel Shift in Fluorescence Microscopy

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In multicolored fluorescence microscopy, pixel shift can cause imaging errors, which can lead to erroneous interpretation of biological data. This article highlights some key considerations in the design of optical filter sets with “zero pixel shift” (less than one-pixel error) performance. Optical filters are typically the only component in an imaging system that changes when a sequential, composite image is created. The major imperfection in optical filters that causes pixel shift is beam deviation created by a nonzero wedge angle (nonparallelism) of either the dichroic beamsplitter and/or the emission filter since both of these filters are in the imaging path. A variation in the parallelism of filters in the filter sets associated with different colors causes the different emission beams to be deflected by varying amounts, thereby producing a pixel shift between the different-colored portions of the composite image.