Quantitative tools in the Multi-image Phasor Analysis (MiPA)
Quantitative tools in the Multi-image Phasor Analysis (MiPA)
复制标题
多图像相量分析 (MiPA) 中的定量工具
DOI：
10.1117/12.2626879
复制
发表时间：
2022
影响因子：
5
通讯作者：
B. Barbieri
中科院分区：
文献类型：
--
作者：
Yuansheng Sun;U. Coskun;S. Liao;B. Barbieri
荧光寿命成像显微镜（FLIM）是一种强大的技术，它提供了一个全新的维度来定量荧光探针，除了它的稳态强度和光谱曲线。由于荧光寿命是每个荧光团的特定参数，其测量对于混合物中荧光团的识别具有重要意义：这为多重成像开辟了新的机会，并在许多应用中提高了成像灵敏度（信噪比）和分辨率。更重要的是，由于探针特有的选择性，FLIM可以提供探针微环境的定量信息（离子、pH、氧含量、局部电场、折射率等）；FLIM是定量FRET的最稳健的方法之一，用于研究活标本中的蛋白质-蛋白质相互作用。如今，交钥匙FLIM仪器是一项成熟的技术，可以从几家公司获得，使该技术很容易获得。然而，用户面临的一个主要挑战仍然是分析和解释FLIM数据。与数字频域FLIM（称为FastFLIM）相结合，相量图方法已被证明是解决FLIM数据分析中许多挑战的有效解决方案。在本报告中，我们描述了几种使用ISS visstavision软件多图像相量分析模块中可用的相量图方法的工具，并举例说明了它们在各种应用中的实用性，包括时间分辨多路复用、NADH成像、STED和FRET。
Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique that provides a totally new dimension to quantitate the fluorescent probe, in addition to its steady-state intensity and spectral profiles. As the fluorescence lifetime is a parameter specific of each fluorophore, its measurement is instrumental in the identification of the fluorophore in a mixture: this opens a new opportunity for multiplex imaging and improves imaging sensitivity (signal-to-noise ratio) and resolution in many applications. More importantly, with peculiar selectivity of probes, FLIM can provide quantitative information of the probe microenvironment (ions, pH, oxygen content, local electrical fields, index of refractions, etc.); FLIM is one of the most robust ways of quantifying FRET for studying protein-protein interactions in live specimens. Today, turn-key FLIM instruments are a mature technology available from several companies, making the technique easily accessible. However, a major challenge for the user is still the analysis and interpretation of FLIM data. In combination with the digital frequency domain FLIM (known as FastFLIM), the phasor plots approach has been demonstrated to be an effective solution to tackle many challenges in FLIM data analysis. In this report, we describe several tools using the phasor plots method available in the ISS VistaVision software multiimage phasor analysis module, and demonstrate their utilities with examples for various applications including time-resolved multiplexing, NADH imaging, STED and FRET.
影响因子：
3.4
作者：
Wright, Belinda K.;Andrews, Laura M.;Gratton, Enrico
通讯作者：
Gratton, Enrico