Multifocal multiphoton microscopy with adaptive optical correction

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

Research output: Chapter in Book/Report/Conference proceedingConference contribution book

13 Citations (Scopus)


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-oxidesemiconductor (CMOS) 1024 pixel single-photon avalanche diode (SPAD) array, opening the way to truly highthroughput FLIM. 

Original languageEnglish
Title of host publicationProceedings of SPIE 8588
Subtitle of host publicationMultiphoton Microscopy in the Biomedical Sciences XIII
Place of PublicationBellingham, Washington
Publication statusPublished - 12 Jun 2013
EventMultiphoton Microscopy in the Biomedical Sciences XIII - San Francisco, CA, United States
Duration: 3 Feb 20135 Feb 2013


ConferenceMultiphoton Microscopy in the Biomedical Sciences XIII
Country/TerritoryUnited States
CitySan Francisco, CA


  • adaptive optics
  • fluorescence lifetime
  • Gerchberg-Saxton algorithm
  • multifocal multiphoton microscopy
  • single photon avalanche diode
  • spatial light modulator


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