Active-illumination microscopy

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. The principle of the feedback is to maintain a fixed detection power, thereby transferring high measurement resolution to weak signals while virtually eliminating the possibility of image saturation. As a result, this technique enhances both the weak-signal relative sensitivity and the dynamic range of a laser scanning optical microscope. We have demonstrated a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. While AIM is applicable to any type of scanning microscope, we have applied it specifically to two-photon microscopy.

TPEF stack of GFP-labeled cleared mouse brain without (left) and with (right) AI. Intensities are displayed in log scale to highlight weak structures and noise. Stacks were acquired with identical exposure times. Total energy delivered to sample was slightly less with AI.
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