Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

• DOI: 10.1038/s41377-021-00536-3
• Publication Date: 2021-05-08T03:33:40Z
• Authors: Kun Chen; Rui Yan; Limin Xiang; Ke Xu
• Keywords: Fluorescence-lifetime imaging microscopy

Abstract The multiplexing capability of fluorescence microscopy is severely limited by the broad spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse local emission spectra notably impede attainable throughput. Here we show that using a single, fixed detection band, through frame-synchronized fast scanning excitation wavelength from white lamp via an acousto-optic tunable filter, up six subcellular targets, labeled common fluorophores substantial overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down ~10 ms). demonstrated quantify abundances different same sample unmixing next enables us devise novel, quantitative schemes for both bi-state Förster resonance energy transfer fluorescent biosensors cells. We thus achieve sensitivities spatiotemporal quantifying mitochondrial matrix pH intracellular macromolecular crowding, further demonstrate, first time, absolute three additional target organelles/proteins elucidate complex, Parkin-mediated mitophagy pathway. Together, provides exceptional opportunities highly multiplexed imaging. prospect acquiring images without need dispersion or care response detector tremendous potential.