22 February 2013 Adaptive nonlinear microscopy for whole tissue imaging
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Abstract
Nonlinear microscopy is capable of imaging biological tissue non-invasively with sub-cellular resolution in three dimensions. For efficient multiphoton signal generation, it is necessary to focus high power, ultra-fast laser pulses into a volume of femtolitres. Aberrations introduced either by the system’s optical setup or the sample under investigation cause a broadening of the diffraction limited focal spot which leads to loss of image intensity and resolution. Adaptive optics provides a means to compensate for these aberrations and is capable of restoring resolution and signal strength when imaging at depth. We describe the use of a micro-electro-mechanical systems (MEMS) deformable membrane mirror in a multiphoton adaptive microscope. The aberration correction is determined in a wavefront sensorless approach by rapidly altering the mirror shape with a random search algorithm until the fluorescence or second harmonic signal intensity is improved. We demonstrate the benefits of wavefront correction in a wide-variety of samples, including urea crystals, convallaria and organotypic tissue cultures. We show how the optimization algorithm can be adjusted, for example by including a bleaching compensation, to allow the user to switch between different imaging modalities, producing a versatile approach to aberration correction.
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M. Caroline Müllenbroich, M. Caroline Müllenbroich, Ewan J. McGhee, Ewan J. McGhee, Amanda J. Wright, Amanda J. Wright, Kurt I. Anderson, Kurt I. Anderson, Keith Mathieson, Keith Mathieson, } "Adaptive nonlinear microscopy for whole tissue imaging", Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 85881X (22 February 2013); doi: 10.1117/12.2006375; https://doi.org/10.1117/12.2006375
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