Michalet X, Ulku A, Smith J T, Bruschini C, Weiss S, Charbon E, Intes X
Department of Chemistry & Biochemistry, University of California at Los Angeles, Los Angeles, California, CA 90095, USA.
AQUA Lab, Ecole Polytechnique Fédérale de Lausanne (EPFL), Neuchâtel, Switzerland.
Proc SPIE Int Soc Opt Eng. 2022 Jan-Feb;11965. doi: 10.1117/12.2607833. Epub 2022 Mar 3.
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.
瑞士SPAD2（SS2）是一款为荧光寿命成像而开发的大规模、宽视场时间选通CMOS单光子雪崩二极管（SPAD）成像仪，其性能最近已在可见光谱范围以及寿命在2.5 - 4纳秒范围内的染料的荧光寿命成像显微镜（FLIM）背景下进行了描述。在此，我们探索其在与小动物成像相关的近红外（NIR）区域的能力，在该区域其灵敏度较低，且典型的近红外荧光染料寿命要短得多（1纳秒或更短）。我们在一个基于紧凑型近红外皮秒脉冲激光器、基于工程扩散器的照明光学器件以及适用于微孔板或小动物成像的近红外优化成像镜头的简单宏观成像装置中开展这项研究。由于不同型号的激光重复频率可能不同，但SS2接受的同步信号频率固定为20兆赫兹，我们首先检查了一种简单的分频方案能够实现针对不同激光重复频率的数据记录。接下来，我们使用不同的时间选通宽度采集数据，包括持续时间长于激光周期的选通，并使用标准的非线性最小二乘法拟合（NLSF）和相量分析对所得数据进行分析。我们表明，SS2所具有的固定同步速率和大的选通宽度（10纳秒及以上）并非准确提取1纳秒范围内的寿命以及区分相近寿命的障碍。总之，SS2以及类似的超大选通SPAD成像仪似乎是用于近红外荧光寿命成像的其他宽视场时间分辨探测器的通用替代方案，包括临床前分子应用。
