Characterization of a time-resolved spectral detector for spectral fluorescence lifetime imaging with the parallel 16-channel FastFLIM and the phasor analysis
Characterization of a time-resolved spectral detector for spectral fluorescence lifetime imaging with the parallel 16-channel FastFLIM and the phasor analysis
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使用并行 16 通道 FastFLIM 和相量分析来表征用于光谱荧光寿命成像的时间分辨光谱探测器
DOI：
10.1117/12.2656271
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Yeh, Hsin-Chih
中科院分区：
文献类型：
--
作者：
Sun, Yuansheng;Nguyen, Trung Duc;Chen, Yuan-I;Coskun, Ulas C.;Liao, Shih-Chu;Yeh, Hsin-Chih
多重荧光检测已成为现代生物传感和成像中不可或缺的工具。尽管已经提出了各种激发/检测光学器件设计和解混方案来实现多路复用检测，但是在每个成像像素处成功区分和量化多个荧光团仍然具有挑战性。最近，荧光寿命成像显微镜（FLIM）相量图分析相结合，已显示出许多优势，比其他多重检测方法。作为荧光分子的固有特性，FLIM测量的荧光寿命不受激发功率或探针丰度的影响，并且可以揭示探针微环境的信息（离子，pH值，氧含量，电信号，折射率等）。此外，FLIM是用于研究蛋白质-蛋白质相互作用的定量FRET的最稳健的方法之一。此外，通过将寿命信息与每个像素处的光谱阅读相结合，光谱FLIM向由常规共焦设置收集的时空信息增加了两个维度，从而产生6维（x，y，z，λ，τ，t）数据集。光谱FLIM方法的主要挑战在于数据采集速度和后处理和分析的复杂性。在这里，我们提出了一种新的时间分辨光谱检测器与并行16通道数字频域FLIM（FastFLIM）读出，快速光谱FLIM数据采集。16通道FastFLIM可以产生无偏光谱FLIM数据进行相量分析，明确区分和定量荧光物种在每个图像像素的独特光谱和寿命特征。我们的光谱FastFLIM方法为监测活标本中的多个动态信号事件提供了新的机会，为复杂的生物系统提供了深入的见解。
Multiplexed fluorescence detection has become an indispensable tool in modern biosensing and imaging. Although a variety of excitation/detection optics designs and unmixing schemes have been proposed to achieve multiplexed detection, successful differentiation and quantification of multiple fluorophores at each imaging pixel is still challenging. Recently, fluorescence lifetime imaging microscopy (FLIM) in combination with the phasor plot analysis has shown many advantages over other multiplexed detection methods. Being an intrinsic property of a fluorescent molecule, fluorescence lifetime measured by FLIM is not biased by excitation power or probe abundance and can reveal information on the probe’s microenvironment (ions, pH, oxygen content, electrical signals, index of refractions, etc.). In addition, FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions. Moreover, by combining lifetime information with a spectral reading at each pixel, spectral FLIM adds two more dimensions to the spatiotemporal information collected by a conventional confocal setup, resulting in a 6-dimenional (x, y, z, λ, τ, t) dataset. The major challenges to the spectral FLIM method lie in the data acquisition speed and the complexity in post processing and analysis. Here, we present a new time-resolved spectral detector with parallel 16-channel digital frequency domain FLIM (FastFLIM) readouts, for fast spectral FLIM data acquisition. The 16-channel FastFLIM can produce unbiased spectral FLIM data for phasor analysis that unambiguously discriminate and quantitate fluorescent species with unique spectral and lifetime features at each image pixel. Our spectral FastFLIM method offers new opportunities for monitoring multiple dynamic signaling events in live specimens, providing insights into complex biological systems.
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影响因子：
3.8
作者：
Fereidouni, Farzad;Reitsma, Keimpe;Gerritsen, Hans C.
通讯作者：
Gerritsen, Hans C.
影响因子：
3.8
作者：
Fereidouni, Farzad;Bader, Arjen N.;Gerritsen, Hans C.
通讯作者：
Gerritsen, Hans C.
多图像相量分析 (MiPA) 中的定量工具
影响因子：
5
作者：
Yuansheng Sun;U. Coskun;S. Liao;B. Barbieri
通讯作者：
B. Barbieri
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DOI：
10.1117/12.2217434
发表时间：
2016
期刊：
The Science of the total environment
影响因子：
--
作者：
Yuansheng Sun;U. Coskun;A. C. Ferreon;B. Barbieri;S. Liao
通讯作者：
S. Liao
影响因子：
48
作者：
Scipioni, Lorenzo;Rossetta, Alessandro;Tedeschi, Giulia;Gratton, Enrico
通讯作者：
Gratton, Enrico