Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy

Abstract

Wide-field time-correlated single photon counting detection techniques, where the position and the arrival time of the photons are recorded simultaneously using a camera, have made some advances recently. The technology and instrumentation used for this approach is employed in areas such as nuclear science, mass spectroscopy and positron emission tomography, but here, we discuss some of the wide-field TCSPC methods, for applications in fluorescence microscopy. We describe work by us and others as presented in the Ulitima fast imaging and tracking conference at the Argonne National Laboratory in September 2018, from phosphorescence lifetime imaging (PLIM) microscopy on the microsecond time scale to fluorescence lifetime imaging ( FLIM) on the nanosecond time scale, and highlight some applications of these techniques.

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

“…Some of the advancements in wide-field FLIM include its implementation with Nipkow disc microscopy for fast 3-D FLIM imaging, 142 wide-field coupled with single plane illumination microscopy for high-resolution 3-D FLIM, 144 TG single photon avalanche diode (SPAD) cameras for phasor-based high speed wide-field FLIM, 145 multifrequency widefield, 146,147 and image gating by pockel cells. 148 Current wide-field FLIM systems are discussed in detail by Suhling and Hirvonen et al 149…”

Fluorescence lifetime imaging with a megapixel SPAD camera and neural network lifetime estimation

“…Wide-field FLIM is typically realised using TCSPC in a system with microchannel plate-based gated optical intensifiers combined with a sensor capable of resolving signal position, such as a Charge-Coupled Device (CCD) camera 22,23 . An emerging alternative to aforementioned intensifier based systems are Single Photon Avalanche Diode (SPAD) arrays manufactured with complementary-metal-oxide semiconductor (CMOS) technology 24 , which can operate with TCSPC [25][26][27][28][29][30][31] or time-gated [32][33][34][35][36][37] acquisition mode.…”

Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation

“…With permission from: W. Becker, The bh TCSPC handbook. 8th edition (2019) available on www.becker-hickl.com Also see ( Schoberer and Botchway, 2014 ; Suhling et al, 2019 ).…”

“…FLIM may be constructed around epifluorescence microscope configuration, confocal single and multiphoton excitation microscopy together with intensified charged coupled device (iCCD) or time-correlated single photon counting (TCSPC). Epifluorescence-FLIM mostly use the iCCD method, although recently a wide array single-photon avalanche photo-diode has been demonstrated) ( Suhling et al, 2019 ). The application of frequency-domain lifetime imaging has also been described ( Lakowicz, 2006 ; Schoberer and Botchway, 2014 ).…”