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Proceedings Paper

Multifocal multiphoton microscopy with adaptive optical correction
Author(s): Simao Coelho; Simon Poland; Nikola Krstajic; David Li; James Monypenny; Richard Walker; David Tyndall; Tony Ng; Robert Henderson; Simon Ameer-Beg
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Paper Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a well established approach for measuring dynamic signalling events inside living cells, including detection of protein-protein interactions. The improvement in optical penetration of infrared light compared with linear excitation due to Rayleigh scattering and low absorption have provided imaging depths of up to 1mm in brain tissue but significant image degradation occurs as samples distort (aberrate) the infrared excitation beam. Multiphoton time-correlated single photon counting (TCSPC) FLIM is a method for obtaining functional, high resolution images of biological structures. In order to achieve good statistical accuracy TCSPC typically requires long acquisition times. We report the development of a multifocal multiphoton microscope (MMM), titled MegaFLI. Beam parallelization performed via a 3D Gerchberg–Saxton (GS) algorithm using a Spatial Light Modulator (SLM), increases TCSPC count rate proportional to the number of beamlets produced. A weighted 3D GS algorithm is employed to improve homogeneity. An added benefit is the implementation of flexible and adaptive optical correction. Adaptive optics performed by means of Zernike polynomials are used to correct for system induced aberrations. Here we present results with significant improvement in throughput obtained using a novel complementary metal-oxide-semiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly high-throughput FLIM.

Paper Details

Date Published: 22 February 2013
PDF: 12 pages
Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 858817 (22 February 2013);
Show Author Affiliations
Simao Coelho, King's College London (United Kingdom)
Simon Poland, King's College London (United Kingdom)
Nikola Krstajic, The Univ. of Edinburgh (United Kingdom)
David Li, Univ. of Sussex (United Kingdom)
James Monypenny, King's College London (United Kingdom)
Richard Walker, The Univ. of Edinburgh (United Kingdom)
David Tyndall, The Univ. of Edinburgh (United Kingdom)
Tony Ng, King's College London (United Kingdom)
Robert Henderson, The Univ. of Edinburgh (United Kingdom)
Simon Ameer-Beg, King's College London (United Kingdom)

Published in SPIE Proceedings Vol. 8588:
Multiphoton Microscopy in the Biomedical Sciences XIII
Ammasi Periasamy; Karsten König; Peter T. C. So, Editor(s)

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