Excitation spectral microscopy for highly multiplexed fluoresce nee imagi ng and quantitative biose rising
作者机构：College of ChemistryUniversity of CaliforniaBerkeleyCAUSA. Division of Molecular Biophysics and Integrated BioimagingLawrence Berkeley National LaboratoryBerkeleyCAUSA. Chan Zuckerberg BiohubSan FranciscoCAUSA
出 版 物：《Light(Science & Applications)》 (光（科学与应用)（英文版）)
年 卷 期：2021年第10卷第6期
页 面：1041-1052页
核心收录：
基 金：the National Institute of General Medical Sciences of the National Institutes of Health(DP2GM132681) the Packard Fellowships for Science and Engineering,and the Bakar Fellows Award,to K.X.K.X.is a Chan Zuckerberg Biohub investigator
主 题：spectral excitation exceptional
摘 要：The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral *** imaging offers potential solutions,yet typical approaches to disperse the local emission spectra notably impede the attain able *** we show that using a sin gle,fixed fluoresc ence emission detection band,through frame-synchronized fast seanning of the excitation wavelength from a white lamp via an acousto-optic tun able filter,up to six subcellular targets,labeled by common fluorophores of substantial spectral overlap,can be simultaneously imaged in live cells with low(〜1%)crosstalks and high temporal resolutions(down to~10 ms).The dem on strated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel,quantitative imaging schemes for both bi-state and Forster resonance energy transfer fluorescent biosensors in live *** thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding,and further demonstrate,for the first time,the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex,Parkin-mediated mitophagy ***,excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence *** prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.
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