Two-photon lifetime-based photoconversion of EGFP for 3D-photostimulation in FLIM.

IF 2.4 3区化学 Q3 CHEMISTRY, ANALYTICAL

Methods and Applications in Fluorescence Pub Date : 2023-06-12 DOI:10.1088/2050-6120/acdb31

Dita Strachotová, Aleš Holoubek, Barbora Brodská, Petr Herman

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Abstract

Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the light intensity allows for an accurate 3D localization of the photoconverted volume within cellular structures, which is especially useful for kinetic FLIM applications. For illustration, we used the two photon photoconversion of EGFP for measurements of redistribution kinetics of nucleophosmin and histone H2B in nuclei of live cells. Measurements revealed high mobility of fluorescently tagged histone H2B in the nucleoplasm and their redistribution between spatially separated nucleoli.

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基于双光子寿命的EGFP在FLIM中用于3d光刺激的光转换。

增强型绿色荧光蛋白(EGFP)是一种广泛应用于细胞和生物医学的荧光标签。令人惊讶的是，EGFP的一些有趣的光化学性质仍未被探索。在这里，我们报道了双光子诱导的EGFP光转化，它可以通过强红外照射永久地转化为具有短荧光寿命和光谱保守发射的形式。因此，光电转换的EGFP可以通过时间分辨检测与未转换的标签区分开来。双光子光转换效率和光强的非线性依赖关系允许在细胞结构中对光转换体积进行精确的3D定位，这对于动力学FLIM应用特别有用。为了说明这一点，我们使用了EGFP的双光子光转换来测量核磷蛋白和组蛋白H2B在活细胞细胞核中的再分配动力学。测量结果显示，荧光标记组蛋白H2B在核质中的高迁移率及其在空间分离的核仁之间的重新分布。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods and Applications in Fluorescence CHEMISTRY, ANALYTICALCHEMISTRY, PHYSICAL&n-CHEMISTRY, PHYSICAL

CiteScore

6.20

自引率

3.10%

发文量

期刊介绍： Methods and Applications in Fluorescence focuses on new developments in fluorescence spectroscopy, imaging, microscopy, fluorescent probes, labels and (nano)materials. It will feature both methods and advanced (bio)applications and accepts original research articles, reviews and technical notes.