HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence

Hsiao Ju Chiang; Daniel E S Koo; Masahiro Kitano; Sean Burkitt; Jay R Unruh; Cristina Zavaleta; Le A Trinh; Scott E Fraser; Francesco Cutrale

doi:10.1038/s41592-022-01751-5

HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal-to-noise fluorescence

Nat Methods. 2023 Feb;20(2):248-258. doi: 10.1038/s41592-022-01751-5. Epub 2023 Jan 19.

Authors

Hsiao Ju Chiang^#^{1

2}, Daniel E S Koo^#^{1

2}, Masahiro Kitano^{1

3}, Sean Burkitt², Jay R Unruh⁴, Cristina Zavaleta², Le A Trinh^{1

3}, Scott E Fraser^{1

2

3}, Francesco Cutrale^{5

6}

Affiliations

¹ Translational Imaging Center, University of Southern California, Los Angeles, CA, USA.
² Department of Biomedical Engineering, University of Southern California, Los Angeles, CA, USA.
³ Molecular and Computational Biology, University of Southern California, Los Angeles, CA, USA.
⁴ Stowers Institute for Medical Research, Kansas City, MO, USA.
⁵ Translational Imaging Center, University of Southern California, Los Angeles, CA, USA. cutrale@usc.edu.
⁶ Department of Biomedical Engineering, University of Southern California, Los Angeles, CA, USA. cutrale@usc.edu.

^# Contributed equally.

Abstract

The expansion of fluorescence bioimaging toward more complex systems and geometries requires analytical tools capable of spanning widely varying timescales and length scales, cleanly separating multiple fluorescent labels and distinguishing these labels from background autofluorescence. Here we meet these challenging objectives for multispectral fluorescence microscopy, combining hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). HyU is efficient and robust, capable of quantitative signal separation even at low illumination levels. In dynamic imaging of developing zebrafish embryos and in mouse tissue, HyU was able to cleanly and efficiently unmix multiple fluorescent labels, even in demanding volumetric timelapse imaging settings. HyU permits high dynamic range imaging, allowing simultaneous imaging of bright exogenous labels and dim endogenous labels. This enables coincident studies of tagged components, cellular behaviors and cellular metabolism within the same specimen, providing more accurate insights into the orchestrated complexity of biological systems.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Animals
Mice
Microscopy, Fluorescence / methods
Zebrafish*