Scientific Results

Adaptive nonlinear microscopy for whole tissue imaging

Year: 2013

Authors: M├╝llenbroich M.C., McGhee E.J., Wright A.J., Anderson K.I., Mathieson K.

Autors Affiliation: Institute of Photonics, Scottish University Physics Alliance, University of Strathclyde, 106 Rottenrow, G4 0NW Glasgow, United Kingdom; Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, G61 1BD, Glasgow, United Kingdom; Institute of Biophysics, Imaging and Optical Science, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom

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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KeyWords: Aberration correction; Deformable membrane mirrors; Nonlinear microscopy; SHG; TPEF, Adaptive optics; Algorithms; MEMS; Tissue; Tissue culture; Ultrafast lasers; Urea; Wavefronts, Fluorescence microscopy
DOI: 10.1117/12.2006375

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