美国加州大学欧文分校Enrico Gratton研究团队开发出一种快速可靠的光谱荧光寿命成像新方法。该项研究成果于2021年4月15日在线发表在《自然—方法学》杂志上。

通过技术和计算的进步，研究人员开发了一个可靠且无偏倚的频谱荧光寿命成像显微技术（S-FLIM）。这个方法（Phasor S-FLIM）将真正的并行多通道数字频域电子学与多维相量方法结合在一起，以光学分辨率提取有关荧光样本光物理特性的详细精确信息。

为了展示Phasor S-FLIM技术的灵活性及其在生物和生物医学领域的应用，研究人员解决了四个常见但具有挑战性的问题：来自多个未知物种的光谱和生命周期特征的盲分解，生物传感器的无偏通透和无背景Förster共振能量转移分析，活细胞中环境敏感探针的光物理特征以及肿瘤球体中的平行四色FLIM成像。

据介绍，FLIM和光谱成像是增加显微技术维度的两种常用方法。但是，它们的组合通常在获取和处理方面效率低下且速度慢。

附：英文原文

Title: Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging

Author: Lorenzo Scipioni, Alessandro Rossetta, Giulia Tedeschi, Enrico Gratton

Issue&Volume: 2021-04-15

Abstract: Fluorescence lifetime imaging microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids. Phasor S-FLIM combines novel electronics for multichannel fluorescence lifetime acquisition and a phasor-based unmixing algorithm for real-time analysis of reliable spectral lifetime imaging data, enabling new biological observations.

DOI: 10.1038/s41592-021-01108-4

Source: https://www.nature.com/articles/s41592-021-01108-4