12 February 2007 Two-­photon fluorescence background rejection by differential aberration imaging
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Abstract
We present a simple and robust way to reject out-of-focus background when performing deep two-photon excited fluorescence (TPEF) imaging in thick tissue. The technique is based on the use of a deformable mirror (DM) to introduce illumination aberrations that preferentially degrade TPEF signal while leaving TPEF background relatively unchanged. A subtraction of aberrated from unaberrated images leads to background rejection. We present a heuristic description of our technique, which we corroborate with experiment. Images of a labeled mouse olfactory bulb are compared with standard TPEF microscopy images, demonstrating significant out of focus TPEF background rejection with our technique. Finally we improve our technique by developing a faster aberration modulation mechanism that performs background subtraction line by line rather than frame by frame. In this manner, the overall image acquisition rate of our technique is the same as that of a standard TPEF microscope.
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Aymeric Leray, Aymeric Leray, Kyle Lillis, Kyle Lillis, Jerome Mertz, Jerome Mertz, } "Two-­photon fluorescence background rejection by differential aberration imaging", Proc. SPIE 6442, Multiphoton Microscopy in the Biomedical Sciences VII, 64421G (12 February 2007); doi: 10.1117/12.701252; https://doi.org/10.1117/12.701252
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