The influence of specimen refractive index, detector signal integration, and non‐uniform scan speed on the imaging properties in confocal microscopy

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Abstract
SUMMARY: Refraction of light in a specimen volume may cause aberrations that influence the imaging properties in confocal microscopy. In this paper the influence on three‐dimensional resolution and geometry is experimentally investigated for a uniform specimen volume. It is found that the depth resolution is more severely affected than the lateral resolution. This is unfortunate, because even under ideal conditions the depth resolution is lower than the lateral resolution. Lateral image geometry is little affected by the specimen refractive index, whereas the depth scale can be considerably elongated or compressed.The influence of a finite detector integration time is also considered. This can give a noticeable, but not particularly severe effect on the image resolution in the line‐scan direction. Because the integration time can be accurately controlled, a shorter integration time can be used when maximum resolution is essential, albeit at the price of a higher noise level.In scanning fluorescence microscopy a non‐uniform scan speed may give large variations in bleaching over the specimen surface. Experiments illustrate how serious such non‐uniform bleaching effects can be when a specimen area is repeatedly scanned, for example when recording optical serial sections.