The performance of SwissSPAD2 (SS2), a large scale, widefield time-gated CMOS SPAD imager developed for fluorescence lifetime imaging, has recently been described in the context of visible range and fluorescence lifetime imaging microscopy (FLIM) of dyes with lifetimes in the 2.5 - 4 ns range. Here, we explore its capabilities in the NIR regime relevant for small animal imaging, where its sensitivity is lower and typical NIR fluorescent dye lifetimes are much shorter (1 ns or less). We carry out this study in a simple macroscopic imaging setup based on a compact NIR picosecond pulsed laser, an engineered diffuser-based illumination optics, and NIR optimized imaging lens suitable for well-plate or small animal imaging. Because laser repetition rates can vary between models, but the synchronization signal frequency accepted by SS2 is fixed to 20 MHz, we first checked that a simple frequency-division scheme enables data recording for different laser repetition rates. Next, we acquired data using different time gate widths, including gates with duration longer than the laser period, and analyzed the resulting data using both standard nonlinear least-square fit (NLSF) and phasor analysis. We show that the fixed synchronization rate and large gate widths characterizing SS2 (10 ns and over) are not an obstacle to accurately extracting lifetime in the 1 ns range and to distinguishing between close lifetimes. In summary, SS2 and similar very large gated SPAD imagers appear as a versatile alternative to other widefield time-resolved detectors for NIR fluorescence lifetime imaging, including preclinical molecular applications.
|Title of host publication||Multiphoton Microscopy in the Biomedical Sciences XXII|
|Editors||Ammasi Periasamy, Peter T. C. So, Karsten Konig, Karsten Konig|
|State||Published - 2022|
|Event||Multiphoton Microscopy in the Biomedical Sciences XXII 2022 - Virtual, Online|
Duration: 20 Feb 2022 → 24 Feb 2022
|Name||Progress in Biomedical Optics and Imaging - Proceedings of SPIE|
|Conference||Multiphoton Microscopy in the Biomedical Sciences XXII 2022|
|Period||20/02/22 → 24/02/22|
Bibliographical noteFunding Information:
This work was supported by the National Institute of Health Grants (R01CA237267, R01CA207725 and R01CA250636), by the UCLA Jonsson Comprehensive Cancer Center Seed Grant Program and in part by the Department of Energy grant DE-SC0020338. A.U. was supported through the Swiss National Science Foundation under grant 200021-166289.
Copyright © 2022 SPIE.
- small animal imaging
- time-gated SPAD imager