Centre for Micro-photonics, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, John Street, PO Box 218, Hawthorn, Victoria, 3122, Australia.
J Fluoresc. 2013 Jul;23(4):671-9. doi: 10.1007/s10895-013-1174-1. Epub 2013 Mar 8.
Fluorescence lifetime imaging microscopy or FLIM provides a versatile tool for spatially-mapping macromolecular interactions and environments through pixel-by-pixel resolution of the excited-state lifetime. In conventional frequency-domain FLIM the phase and modulation of the detected fluorescence are determined by the photophysics of the fluorophore only. However, translational motion on the timescale of FLIM acquisition can significantly perturb apparent phase and modulation values owing to intensity fluctuations and phase decoherence. Using the phasor plot we outline a simple analytic theory, numerical simulations and measurements on fluorescent beads (ex 470 nm, em 520 nm). Fluctuations due to particle motions result in an increase in the number and spread of phasors, an effect we refer to as phasor broadening. The approach paves the way for the measurement of lifetimes and translational motion from one experiment.
荧光寿命成像显微镜（FLIM）通过激发态寿命的逐像素分辨率，为空间映射生物大分子相互作用和环境提供了一种通用工具。在传统的频域 FLIM 中，检测荧光的相位和调制仅由荧光团的光物理决定。然而，在 FLIM 采集的时间尺度上的平移运动由于强度波动和相位去相干，会显著干扰明显的相位和调制值。使用相图，我们概述了一种简单的分析理论、数值模拟和对荧光珠的测量（激发 470nm，发射 520nm）。由于粒子运动引起的波动导致相矢量数量和分布的增加，我们称之为相矢量展宽。这种方法为从一次实验中测量寿命和平移运动铺平了道路。
