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Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing
- Source :
- Light, science & applications, vol 10, iss 1, Light, Science & Applications, Light: Science & Applications, Vol 10, Iss 1, Pp 1-12 (2021)
- Publication Year :
- 2022
- Publisher :
- SPIE, 2022.
-
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.<br />Spectrally resolved fluorescence microscopy is achieved through frame-synchronized scanning of the excitation wavelength, thus enabling highly multiplexed fast imaging of live cells for both multitargets and quantitative biosensing.
- Subjects :
- medicine.medical_specialty
Fluorescence-lifetime imaging microscopy
Materials science
Bioengineering
Optical Physics
01 natural sciences
Article
Fluorescence spectroscopy
010309 optics
03 medical and health sciences
0103 physical sciences
Spectral width
Microscopy
Fluorescence microscope
medicine
Applied optics. Photonics
030304 developmental biology
0303 health sciences
business.industry
QC350-467
Optics. Light
Fluorescence
Atomic and Molecular Physics, and Optics
TA1501-1820
Electronic, Optical and Magnetic Materials
Spectral imaging
Förster resonance energy transfer
Optoelectronics
business
Biotechnology
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- High-Speed Biomedical Imaging and Spectroscopy VII
- Accession number :
- edsair.doi.dedup.....c41002297f712f80a953a565a91ccebb