Wang P, Hecht F, Ossato G, Tille S, Fraser S E, Junge J A
Translational Imaging Center, Dornsife School of Letters, Arts, and Sciences, University of Southern California, 1002 Childs Way, Los Angeles, CA 90089, USA.
Leica Microsystems CMS GmbH, Am Friedensplatz 3, Mannheim 68165, Germany.
Biomed Opt Express. 2021 May 17;12(6):3463-3473. doi: 10.1364/BOE.420953. eCollection 2021 Jun 1.
Fluorescence lifetime imaging microscopy (FLIM) with phasor analysis provides easy visualization and analysis of fluorophores' lifetimes which is valuable for multiple applications including metabolic imaging, STED imaging, FRET imaging and functional imaging. However, FLIM imaging typically suffers from low photon budgets, leading to unfavorable signal to noise ratios which in many cases prevent extraction of information from the data. Traditionally, median filters are applied in phasor analysis to tackle this problem. This unfortunately degrades high spatial frequency FLIM information in the phasor analysis. These high spatial frequency components are typically edges of features and puncta, which applies to membranes, mitochondria, granules and small organelles in a biological sample. To tackle this problem, we propose a filtering strategy with complex wavelet filtering and Anscombe transform for FLIM phasor analysis. This filtering strategy preserves fine structures and reports accurate lifetimes in photon starved FLIM imaging. Moreover, this filter outperforms median filters and makes FLIM imaging with lower laser power and faster imaging possible.
采用相量分析的荧光寿命成像显微镜（FLIM）能够轻松实现对荧光团寿命的可视化和分析，这对于包括代谢成像、受激发射损耗（STED）成像、荧光共振能量转移（FRET）成像和功能成像在内的多种应用都具有重要价值。然而，FLIM成像通常存在光子预算低的问题，导致信噪比不理想，在许多情况下阻碍了从数据中提取信息。传统上，在相量分析中应用中值滤波器来解决这个问题。不幸的是，这会降低相量分析中高空间频率的FLIM信息。这些高空间频率成分通常是特征和点的边缘，适用于生物样本中的膜、线粒体、颗粒和小细胞器。为了解决这个问题，我们提出了一种用于FLIM相量分析的复杂小波滤波和安斯库姆变换的滤波策略。这种滤波策略能够保留精细结构，并在光子匮乏的FLIM成像中报告准确的寿命。此外，这种滤波器优于中值滤波器，使得以更低的激光功率和更快的成像速度进行FLIM成像成为可能。
