Multifocal multiphoton microscopy with adaptive optical correction

Simao Coelho; Simon Poland; Nikola Krstajic; David Li; James Monypenny; Richard Walker; David Tyndall; Tony Ng; Robert Henderson; Simon Ameer-Beg

doi:10.1117/12.2000188

22 February 2013 Multifocal multiphoton microscopy with adaptive optical correction

Simao Coelho, Simon Poland, Nikola Krstajic, David Li, James Monypenny, Richard Walker, David Tyndall, Tony Ng, Robert Henderson, Simon Ameer-Beg

Author Affiliations +

Proceedings Volume 8588, Multiphoton Microscopy in the Biomedical Sciences XIII; 858817 (2013) https://doi.org/10.1117/12.2000188
Event: SPIE BiOS, 2013, San Francisco, California, United States

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.

Citation Download Citation

Simao Coelho, Simon Poland, Nikola Krstajic, David Li, James Monypenny, Richard Walker, David Tyndall, Tony Ng, Robert Henderson, and Simon Ameer-Beg "Multifocal multiphoton microscopy with adaptive optical correction", Proc. SPIE 8588, Multiphoton Microscopy in the Biomedical Sciences XIII, 858817 (22 February 2013); https://doi.org/10.1117/12.2000188

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available