New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime

Poland, Simon P. and Erdogan, Ahmet T. and Krstajic, Nikola and Levitt, James and Devauges, Viviane and Walker, Richard J. and Li, David Day-Uei and Ameer-Beg, Simon M. and Henderson, Robert K. (2016) New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime. Optics Express, 24 (7). pp. 6899-6915. ISSN 1094-4087 (https://doi.org/10.1364/OE.24.006899)

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Abstract

We demonstrate an implementation of a centre-of-mass method (CMM) incorporating background subtraction for use in multifocal fluorescence lifetime imaging microscopy to accurately determine fluorescence lifetime in live cell imaging using the Megaframe camera. The inclusion of background subtraction solves one of the major issues associated with centre-of-mass approaches, namely the sensitivity of the algorithm to background signal. The algorithm, which is predominantly implemented in hardware, provides real-time lifetime output and allows the user to effectively condense large amounts of photon data. Instead of requiring the transfer of thousands of photon arrival times, the lifetime is simply represented by one value which allows the system to collect data up to limit of pulse pile-up without any limitations on data transfer rates. In order to evaluate the performance of this new CMM algorithm with existing techniques (i.e. Rapid lifetime determination and Levenburg-Marquardt), we imaged live MCF-7 human breast carcinoma cells transiently transfected with FRET standards. We show that, it offers significant advantages in terms of lifetime accuracy and insensitivity to variability in dark count rate (DCR) between Megaframe camera pixels. Unlike other algorithms no prior knowledge of the expected lifetime is required to perform lifetime determination. The ability of this technique to provide real-time lifetime readout makes it extremely useful for a number of applications.

  • Item type:Article
    ID code:55799
    Dates:
    Date
    Event
    4 April 2016
    Published
    21 March 2016
    Published Online
    7 March 2016
    Accepted
    Notes:© 2016 Optical Society of America. One print or electronic copy may be made for personal use only. Systematic reproduction and distribution, duplication of any material in this paper for a fee or for commercial purposes, or modifications of the content of this paper are prohibited.
    Subjects:Science > Physics > Optics. Light
    Department:Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences
    Depositing user: Pure Administrator
    Date deposited:08 Mar 2016 11:51
    Last modified:14 Apr 2024 00:35
    Related URLs:
    URI:https://strathprints.strath.ac.uk/id/eprint/55799
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