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

Simon P. Poland, Ahmet T. Erdogan, Nikola Krstajic, James Levitt, Viviane Devauges, Richard J. Walker, David Day-Uei Li, Simon M. Ameer-Beg, Robert K. Henderson

Research output: Contribution to journalArticle

16 Citations (Scopus)
39 Downloads (Pure)

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.
Original languageEnglish
Pages (from-to)6899-6915
Number of pages17
JournalOptics Express
Volume24
Issue number7
Early online date21 Mar 2016
DOIs
Publication statusPublished - 4 Apr 2016

Keywords

  • centre of mass method
  • fluorescence lifetime
  • imaging
  • microscopy

Projects

Supply of Firmware to KCL

Li, D.

King's College London

Project: Knowledge Exchange (Services/Consultancy)

Smart solid-state Raman spectrometers

Li, D. & Lin, S.

Royal Society

16/03/1515/09/17

Project: Research

Cite this

Poland, S. P., Erdogan, A. T., Krstajic, N., Levitt, J., Devauges, V., Walker, R. J., Li, D. D-U., Ameer-Beg, S. M., & Henderson, R. K. (2016). New high-speed centre of mass method incorporating background subtraction for accurate determination of fluorescence lifetime. Optics Express, 24(7), 6899-6915. https://doi.org/10.1364/OE.24.006899