Development of a fast TCSPC FLIM-FRET imaging system

Simon P. Poland, Simao Coelho, Nikola Krstajić, David Tyndall, Richard Walker, James Monypenny, David Day Uei Li, Robert Henderson, Simon Ameer-Beg

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

    15 Citations (Scopus)

    Abstract

    Forster/Fluorescence resonant energy transfer (FRET) has become an extremely important technique to explore biological interactions in cells and tissues. As the non-radiative transfer of energy from the donor to acceptor occurs typically only within 1-10nm, FRET measurement allows the user to detect localisation events between proteinconjugated fluorophores. Compared to other techniques, the use of time correlated single photon counting (TCSPC) to measure fluorescence lifetime (FLIM) has become the gold standard for measuring FRET interactions in cells. The technique is fundamentally superior to all existing techniques due to its near ideal counting efficiency, inherent low excitation light flux (reduced photobleaching and toxicity) and time resolution. Unfortunately due to its slow acquisition time when compared with other techniques, such as Frequency-domain lifetime determination or anisotropy, this makes it impractical for measuring dynamic protein interactions in cells. The relatively slow acquisition time of TCSPC FLIMFRET is simply due to the system usually employing a single-beam scanning approach where each lifetime (and thus FRET interaction) is determined individually on a voxel by voxel basis. In this paper we will discuss the development a microscope system which will parallelize TCSPC for FLIM-FRET in a multi-beam multi-detector format. This will greatly improve the speed at which the system can operate, whilst maintaining both the high temporal resolution and the high signal-to-noise for which typical TCPSC systems are known for. We demonstrate this idea using spatial light modulator (SLM) generated beamlets and single photon avalanche detector (SPAD) array. The performance is evaluated on a plant specimen.

    Original languageEnglish
    Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XIII
    PublisherSPIE-International Society for Optical Engineering
    Number of pages8
    Volume8588
    ISBN (Print)9780819493576
    DOIs
    Publication statusPublished - 2013
    EventMultiphoton Microscopy in the Biomedical Sciences XIII - San Francisco, CA, United States
    Duration: 3 Feb 20135 Feb 2013

    Conference

    ConferenceMultiphoton Microscopy in the Biomedical Sciences XIII
    CountryUnited States
    CitySan Francisco, CA
    Period3/02/135/02/13

    Keywords

    • FLIM-FRET
    • Fluorescence lifetime
    • In-vivo imaging
    • Multifocal scanning
    • Multiphoton microscopy
    • TCSPC

    Fingerprint Dive into the research topics of 'Development of a fast TCSPC FLIM-FRET imaging system'. Together they form a unique fingerprint.

  • Cite this

    Poland, S. P., Coelho, S., Krstajić, N., Tyndall, D., Walker, R., Monypenny, J., Li, D. D. U., Henderson, R., & Ameer-Beg, S. (2013). Development of a fast TCSPC FLIM-FRET imaging system. In Multiphoton Microscopy in the Biomedical Sciences XIII (Vol. 8588). [85880X] SPIE-International Society for Optical Engineering. https://doi.org/10.1117/12.2004199