Adaptive Optimisation of Illumination Beam Profiles in Fluorescence Microscopy

Mitchell, T.J., Saunter, C.D., O'Nions, W., Girkin, J.M. and Love, G.D. (2015) Adaptive Optimisation of Illumination Beam Profiles in Fluorescence Microscopy. In: Adaptive Optics and Wavefront Control for Biological Systems, San Francisco, CA, USA, 07-09 Feb 2015, 93350B. ISBN 9781628414257 (doi: 10.1117/12.2080310)

Abstract

Wide-field fluorescence microscope techniques such as single/selective plane illumination microscope (SPIM) are typically configured to image large regions of a sample at once. Here the illumination beam provides uniform excitation of several biological features across the region, `sliced' to a thickness of between 5-10 microns. In this paper we propose a simple alteration to the optical configuration of a SPIM by switching the light-sheet- forming cylindrical lens with a spatial light modulator. This has the potential to adaptively reconfigure the light sheet geometry to improve the optical sectioning of specific biological features, rather than the thicker sectioning of several features at once across a larger observation field-of-view. We present a prototype version of such a system, referred to as an Adaptive-SPIM (A-SPIM) system. We then suggest that the direct recording of illumination beam shapes within the working microscope system can better facilitate the analysis and subsequent re-configuration of the illumination beam to a specific geometry, and summarise the design and operation of a device that we have developed specifically for this purpose. We finally present reconstructed quantitative three dimensional flux maps of illumination beams from three microscope configurations taken using this miniature high-dynamic range beam profiling device, comparing the beam geometry of a regular SPIM system with our prototype A-SPIM system, and suggesting future improvements.

• Item Type: Conference Proceedings
• Keywords: Fluorescence microscopy, SPIM, embryogenesis, beam shaping, beam diagnostics.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Mitchell, Dr Thomas
• Authors: Mitchell, T.J., Saunter, C.D., O'Nions, W., Girkin, J.M., and Love, G.D.
• College/School: College of Science and Engineering > School of Physics and Astronomy
• Journal Name: Proc. SPIE
• ISSN: 1605-7422
• ISBN: 9781628414257
• Copyright Holders: Copyright © 2015 SPIE
• First Published: First published in Proceedings of SPIE 9335: 93350B
• Publisher Policy: Reproduced in accordance with the publisher copyright policy