Weidtkamp-Peters Stefanie, Felekyan Suren, Bleckmann Andrea, Simon Rüdiger, Becker Wolfgang, Kühnemuth Ralf, Seidel Claus A M
Lehrstuhl für Molekulare Physikalische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225, Düsseldorf, Germany.
Photochem Photobiol Sci. 2009 Apr;8(4):470-80. doi: 10.1039/b903245m. Epub 2009 Mar 6.
Multiparameter Fluorescence Image Spectroscopy (MFIS) is used to monitor simultaneously a variety of fluorescence parameters in confocal fluorescence microscopy. As the photons are registered one by one, MFIS allows for fully parallel recording of Fluorescence Correlation/Cross Correlation Spectroscopy (FCS/FCCS), fluorescence lifetime and pixel/image information over time periods of hours with picosecond accuracy. The analysis of the pixel fluorescence information in higher-dimensional histograms maximizes the selectivity of fluorescence microscopic methods. Moreover it facilitates a statistically-relevant data analysis of the pixel information which makes an efficient detection of heterogeneities possible. The reliability of MFIS has been demonstrated for molecular interaction studies in different complex environments: (I) detecting the heterogeneity of diffusion properties of the dye Rhodamine 110 in a sepharose bead, (II) Förster Resonance Energy Transfer (FRET) studies in mammalian HEK293 cells, and (III) FRET study of the homodimerisation of the transcription factor BIM1 in plant cells. The multidimensional analysis of correlated changes of several parameters measured by FRET, FCS, fluorescence lifetime and anisotropy increases the robustness of the analysis significantly. The economic use of photon information allows one to keep the expression levels of fluorescent protein-fusion proteins as low as possible (down to the single-molecule level).
多参数荧光图像光谱技术（MFIS）用于在共聚焦荧光显微镜中同时监测多种荧光参数。由于光子是逐个记录的，MFIS允许以皮秒精度在数小时的时间段内对荧光相关/交叉相关光谱（FCS/FCCS）、荧光寿命和像素/图像信息进行完全并行记录。在高维直方图中对像素荧光信息进行分析可最大限度地提高荧光显微镜方法的选择性。此外，它有助于对像素信息进行具有统计学意义的数据分析，从而能够有效地检测异质性。MFIS的可靠性已在不同复杂环境中的分子相互作用研究中得到证明：（I）检测琼脂糖珠中罗丹明110染料扩散特性的异质性，（II）在哺乳动物HEK293细胞中进行的荧光共振能量转移（FRET）研究，以及（III）植物细胞中转录因子BIM1同二聚化的FRET研究。通过对FRET、FCS、荧光寿命和各向异性测量的几个参数的相关变化进行多维分析，可显著提高分析的稳健性。光子信息的经济利用使荧光蛋白融合蛋白的表达水平尽可能低（低至单分子水平）。
