Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent
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.
more » « less- NSF-PAR ID:
- 10392094
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Methods
- Volume:
- 20
- Issue:
- 2
- ISSN:
- 1548-7091
- Page Range / eLocation ID:
- p. 248-258
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Availability and implementation The source code used for this paper was written in Julia and is available with the test data at https://github.com/brossetti/ssasu.
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