Kim Dongeun, Moon Sucbei, Hwang Wonsang, Young Kim Dug
Opt Express. 2022 Apr 25;30(9):14677-14685. doi: 10.1364/OE.450761.
We investigated the possibility of using long excitation pulses in fluorescence lifetime imaging microscopy (FLIM) using phasor analysis. It has long been believed that the pulse width of an excitation laser must be shorter than the lifetime of a fluorophore in a time-domain FLIM system. Even though phasor analysis can effectively minimize the pulse effect by using deconvolution, the precision of a measured lifetime can be degraded seriously. Here, we provide a fundamental theory on pulse-width-dependent measurement precisions in lifetime measurement in the phasor plane. Our theory predicts that high-precision lifetimes can be obtained even with a laser whose pulse width is four times larger than the lifetime of a fluorophore. We have experimentally demonstrated this by measuring the lifetimes of fluorescence probes with 2.57 ns and 3.75 ns lifetimes by using various pulse widths (0.52-38 ns) and modulation frequencies (10-200 MHz). We believe our results open a new possibility of using long pulse-width lasers for high-precision FLIM.
我们利用相量分析研究了在荧光寿命成像显微镜（FLIM）中使用长激发脉冲的可能性。长期以来，人们一直认为，在时域FLIM系统中，激发激光的脉冲宽度必须短于荧光团的寿命。尽管相量分析可以通过去卷积有效地最小化脉冲效应，但测量寿命的精度仍可能严重下降。在此，我们提供了相量平面中寿命测量中与脉冲宽度相关的测量精度的基本理论。我们的理论预测，即使使用脉冲宽度比荧光团寿命大四倍的激光，也能获得高精度的寿命。我们通过使用各种脉冲宽度（0.52 - 38 ns）和调制频率（10 - 200 MHz）测量寿命分别为2.57 ns和3.75 ns的荧光探针的寿命，从实验上证明了这一点。我们相信我们的结果为使用长脉冲宽度激光进行高精度FLIM开辟了新的可能性。
