Adaptive nonlinear microscopy for whole tissue imaging

Müllenbroich, M. C. , McGhee, E. J., Wright, A. J., Anderson, K. I. and Mathieson, K. (2013) Adaptive nonlinear microscopy for whole tissue imaging. In: Ammasi, P., Konig, K. and So, P.T.C. (eds.) Multiphoton Microscopy in the Biomedical Sciences XIII. Series: SPIE proceedings (8588). SPIE. ISBN 9780819493576 (doi:10.1117/12.2006375)

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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.

Item Type:Book Sections
Glasgow Author(s) Enlighten ID:Muellenbroich, Dr Caroline
Authors: Müllenbroich, M. C., McGhee, E. J., Wright, A. J., Anderson, K. I., and Mathieson, K.
College/School:College of Science and Engineering > School of Physics and Astronomy

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