Time synchronization of LED excitation source and dual-camera for quantitative FRET imaging

IF 2.2 3区工程技术 Q1 MICROSCOPY

Micron Pub Date : 2025-07-14 DOI:10.1016/j.micron.2025.103870

Hai Wen , Huan Zuo , Xinghong Cai, Xinlei Li, Zhengfei Zhuang, Tongsheng Chen

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引用次数: 0

Abstract

Multi-wavelength LED light source has been widely used as excitation source in fluorescence microscopy. This study presents a six-wavelength LED excitation source that integrates an internal time synchronization control mechanism, enabling high-precision synchronization between LED excitation and dual-camera imaging without requiring additional control hardware. The control system is built around an STM32F429 microcontroller and employs a hardware-triggered synchronization strategy specifically designed for quantitative fluorescence resonance energy transfer (FRET) imaging. By incorporating this excitation source into a dual-channel fluorescence microscope, we minimized the excitation-exposure timing discrepancy to

10 μ s

, effectively reducing unnecessary exposure and mitigating photobleaching. To assess system performance, we conducted quantitative FRET analysis in living cells expressing different standard FRET plasmids. Experimental results demonstrated that during time-lapse FRET analysis, the synchronization system reduced photobleaching-induced deviations in FRET efficiency (

E_{D}

) and acceptor-to-donor ratio (

R_{c}

) from 18.8% and 15.8% to 5.6% and 2.6%, respectively, thereby enhancing the reliability of FRET quantification. As FRET is widely used to study protein interactions, conformational changes, and signal transduction pathways, this system improves the accuracy of dynamic molecular measurements in live-cell imaging.

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用于定量FRET成像的LED激励源和双相机时间同步

多波长LED光源作为激发光源在荧光显微镜中得到了广泛的应用。本研究提出了一种集成了内部时间同步控制机制的六波长LED激发源，无需额外的控制硬件，即可实现LED激发和双摄像头成像之间的高精度同步。控制系统围绕STM32F429微控制器构建，采用硬件触发同步策略，专为定量荧光共振能量转移（FRET）成像而设计。通过将该激发源与双通道荧光显微镜结合，我们将激发-曝光时间差异减小到10μs，有效地减少了不必要的曝光，减轻了光漂白。为了评估系统性能，我们在表达不同标准FRET质粒的活细胞中进行了定量FRET分析。实验结果表明，在延时FRET分析中，同步系统将光漂白引起的FRET效率（ED）和受体供体比（Rc）的偏差分别从18.8%和15.8%降低到5.6%和2.6%，从而提高了FRET定量的可靠性。由于FRET被广泛用于研究蛋白质相互作用、构象变化和信号转导途径，该系统提高了活细胞成像中动态分子测量的准确性。

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

Micron 工程技术-显微镜技术

CiteScore

4.30

自引率

4.20%

发文量

100

审稿时长

31 days

期刊介绍： Micron is an interdisciplinary forum for all work that involves new applications of microscopy or where advanced microscopy plays a central role. The journal will publish on the design, methods, application, practice or theory of microscopy and microanalysis, including reports on optical, electron-beam, X-ray microtomography, and scanning-probe systems. It also aims at the regular publication of review papers, short communications, as well as thematic issues on contemporary developments in microscopy and microanalysis. The journal embraces original research in which microscopy has contributed significantly to knowledge in biology, life science, nanoscience and nanotechnology, materials science and engineering.