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To minimize photobleaching during acquisition these parameters should be adjusted:
- decreasing the pixel resolution by zooming out or by lowering the pixel number (e.g. 128x128 instead of 512x512)
- decreasing the pixel dwell time using a faster scan speed (this is also preferable to monitor rapid recovery kinetics)
- decreasing the laser power during image acquisition to a minimum
- using fluorophores which are less susceptible to photobleaching at low laser intensities
- frame or line averaging should be avoided to reduce undesired photobleaching in the imaging mode
- opening the pinhole leads to a brighter signal with less laser power
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