Cornelia Wetzker, Marcelo Leomil Zoccoler, Svetlana Iarovenko, Chukwuebuka William Okafornta, Anja Nobst, Hella Hartmann, Thomas Müller-Reichert, Robert Haase, Gunar Fabig
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引用次数: 0
Abstract
Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as an additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime separation, in live-cell imaging using open-source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we separated the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six-dimensional datasets to investigate cell division in the developing early embryo. Second, we separated fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open-source plugin napari-flim-phasor-plotter to implement conversion, visualisation, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.
荧光寿命成像显微镜(FLIM)将荧光团激发态的持续时间转换为寿命信息,作为生物样品图像中对比度的额外来源。这提供了分离荧光团的可能性,在类似激发光谱的情况下特别有益。在这里,我们展示基于FLIM相量分析的荧光分子的区别,称为寿命分离,在活细胞成像中使用开源软件进行分析。我们展示了使用秀丽隐杆线虫作为模型系统的两个应用程序。首先,我们分离了光谱高度重叠的荧光团mCherry和mKate2,在六维数据集中特异性地跟踪标记蛋白,以研究发育中的早期胚胎的细胞分裂。其次,我们在成年雌雄同体中分离了感兴趣的标记蛋白的荧光,从掩盖自然自身荧光中分离出来。在FLIM数据处理和工作流实现方面,我们开发了开源插件napari- film -phasor-plotter,实现了不同格式FLIM数据的转换、可视化、分析和重用。因此,我们的工作促进了FLIM显微镜在生命科学研究中的技术应用和生物图像数据管理和分析。
期刊介绍:
The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit.
The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens.
Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.