Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

Kun Chen; Rui Yan; Limin Xiang; Ke Xu

doi:10.1038/s41377-021-00536-3

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Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

Articles

- Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing
- Light: Science & Applications Vol. 10, Issue 6, Pages: 1041-1052(2021)
- Affiliations：
  
  1.College of Chemistry, University of California, Berkeley, CA, USA
  2.Division of Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
  3.Chan Zuckerberg Biohub, San Francisco, CA, USA
- Author bio：
  
  Ke Xu (xuk@berkeley.edu)
- Funds：
- DOI：10.1038/s41377-021-00536-3
  CLC：
- Published：30 June 2021，
  
  Published Online：08 May 2021，
  
  Received：29 December 2020，
  
  Revised：13 April 2021，
  
  Accepted：15 April 2021
- Accepted：
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Chen, K. et al. Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing. Light: Science & Applications, 10, 1041-1052 (2021).
DOI：

Chen, K. et al. Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing. Light: Science & Applications, 10, 1041-1052 (2021). DOI： 10.1038/s41377-021-00536-3.

摘要

Abstract

The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions

yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single

fixed fluorescence emission detection band

through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable 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 demonstrated 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 Förster resonance energy transfer fluorescent biosensors in live cells. We 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 pathway. Together

excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.

关键词

Keywords

references

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