Single-Photon, Time-Gated, Phasor-Based Fluorescence Lifetime Imaging through Highly Scattering Medium.
Single-Photon, Time-Gated, Phasor-Based Fluorescence Lifetime Imaging through Highly Scattering Medium.
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DOI：
10.1021/acsphotonics.9b00874
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发表时间：
2020-01-15
期刊：
影响因子：
7
通讯作者：
Michalet, Xavier
中科院分区：
文献类型：
--
作者：
Ankri, Rinat;Basu, Arkaprabha;Ulku, Arin Can;Bruschini, Claudio;Charbon, Edoardo;Weiss, Shimon;Michalet, Xavier
关键词：
荧光寿命成像（FLI）越来越被认为是生物化学和细胞研究的有力工具，包括在体内的应用。荧光寿命是任何荧光染料的固有特性，在很大程度上不依赖于激发强度和信号电平。特别是，它允许区分染料与相似的发射光谱，提供额外的多路复用能力。然而，体内可见范围内的FLI由于(i)组织自身荧光的污染而变得复杂，这降低了对比度，以及（ii）组织中的光散射和吸收，这显著降低了荧光强度并改变了信号的时间分布。在这里，我们展示了如何解释和克服这些问题，使用新的时间门控单光子雪崩二极管阵列相机SwissSPAD2，结合相量分析，为终身成像提供了一种简单快速的视觉方法。特别是，我们展示了相量色散是如何随着散射的增加和/或荧光强度的降低而增加的。接下来，我们表明，只要感兴趣的荧光信号大于幻体自身荧光，存在明显的寿命可以通过适当的背景校正清楚地识别。我们利用这些结果证明了通过高散射和自荧光幻影层检测表达荧光蛋白mCyRFP1的A459细胞。这些结果展示了在具有挑战性的条件下使用标准的、明亮的、可见的荧光团或荧光蛋白进行FLI的可能性。
Fluorescence lifetime imaging (FLI) is increasingly recognized as a powerful tool for biochemical and cellular investigations, including in vivo applications. Fluorescence lifetime is an intrinsic characteristic of any fluorescent dye which, to a large extent, does not depend on excitation intensity and signal level. In particular, it allows distinguishing dyes with similar emission spectra, offering additional multiplexing capabilities. However, in vivo FLI in the visible range is complicated by the contamination by (i) tissue autofluorescence, which decreases contrast, and by (ii) light scattering and absorption in tissues, which significantly reduce fluorescence intensity and modify the temporal profile of the signal. Here, we demonstrate how these issues can be accounted for and overcome, using a new time-gated single-photon avalanche diode array camera, SwissSPAD2, combined with phasor analysis to provide a simple and fast visual method for lifetime imaging. In particular, we show how phasor dispersion increases with increasing scattering and/or decreasing fluorescence intensity. Next, we show that as long as the fluorescence signal of interest is larger than the phantom autofluorescence, the presence of a distinct lifetime can be clearly identified with appropriate background correction. We use these results to demonstrate the detection of A459 cells expressing the fluorescent protein mCyRFP1 through highly scattering and autofluorescent phantom layers. These results showcase the possibility to perform FLI in challenging conditions, using standard, bright, visible fluorophore or fluorescence proteins.
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