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Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging.

Francesco Cutrale1,2, Vikas Trivedi1,2,3,4, Le A Trinh1,2

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This study introduces Hyper-Spectral Phasors (HySP) software to improve biological imaging. HySP effectively denoises and unmixes multiple fluorophores in live samples, enhancing imaging quality and speed.

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Area of Science:

  • Biological imaging
  • Microscopy
  • Biophotonics

Background:

  • Time-lapse imaging of multiple labels is difficult due to noise, photobleaching, and phototoxicity.
  • These factors compromise signal quality and limit imaging throughput.
  • Processing time is a major bottleneck in analyzing complex biological samples.

Purpose of the Study:

  • To develop advanced software for denoising and unmixing spectrally overlapping fluorophores.
  • To enable high-throughput, high-quality multi-label time-lapse imaging.
  • To address the challenges of low signal-to-noise ratios in biological imaging.

Main Methods:

  • Development of Hyper-Spectral Phasors (HySP) software.
  • Application of HySP for spectral unmixing and denoising.
  • Utilizing phasor analysis for rapid signal separation.

Main Results:

  • HySP successfully denoises images and unmixes multiple spectrally overlapping fluorophores.
  • The software operates effectively in a low signal-to-noise regime.
  • Achieved unmixing of seven distinct signals in time-lapse imaging of zebrafish embryos.

Conclusions:

  • HySP software significantly enhances the quality and speed of multi-label time-lapse imaging.
  • This tool overcomes critical limitations in current biological imaging techniques.
  • Enables advanced live-cell imaging and analysis of complex biological processes.