Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging

Cutrale, Francesco and Trivedi, Vikas and Trinh, Le A. and Chiu, Chi-Li and Choi, John M. and Artiga, Marcela S. and Fraser, Scott E. (2017) Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging. Nature Methods, 14 (2). pp. 149-152. ISSN 1548-7091. https://resolver.caltech.edu/CaltechAUTHORS:20170117-091624181

	Image (JPEG) (Supplementary Figure 1: Comparison of HySP and linear unmixing under different signal-to-noise ratios (SNRs)) - Supplemental Material See Usage Policy. 850Kb
	Image (JPEG) (Supplementary Figure 2: Errors on hyper-spectral phasor plot) - Supplemental Material See Usage Policy. 228Kb
	Image (JPEG) (Supplementary Figure 3: Sensitivity of phasor point) - Supplemental Material See Usage Policy. 275Kb
	Image (JPEG) (Supplementary Figure 4: Effect of phasor space denoising on scatter error and shifted-mean error) - Supplemental Material See Usage Policy. 611Kb
	Image (JPEG) (Supplementary Figure 5: Effect of phasor space denoising on image intensity) - Supplemental Material See Usage Policy. 970Kb
	Image (JPEG) (Supplementary Figure 6: Phasor analysis for unmixing hyper-spectral fluorescent signals in vivo) - Supplemental Material See Usage Policy. 878Kb
	Image (JPEG) (Supplementary Figure 7: Autofluorescence identification and removal in phasor space) - Supplemental Material See Usage Policy. 927Kb
	Image (JPEG) (Supplementary Figure 8: Optical separation of eGFP and citrine) - Supplemental Material See Usage Policy. 522Kb
	Image (JPEG) (Supplementary Figure 9: Comparison of HySP and linear unmixing in resolving 7 fluorescent signals) - Supplemental Material See Usage Policy. 778Kb
	Image (JPEG) (Supplementary Figure 10: Effect of binning on HySP analysis of 7 in vivo fluorescent signals) - Supplemental Material See Usage Policy. 1051Kb
	Video (MPEG) (Video 1: Truecolor rendering of raw data zebrafish with 2 fluorescent and 1 auto-fluorescent labels) - Supplemental Material See Usage Policy. 6Mb
	Video (MPEG) (Video 2: HySP resolved 2-color zebrafish with autofluorescence removal) - Supplemental Material See Usage Policy. 10Mb
	Video (MPEG) (Video 3: Truecolor rendering of raw data zebrafish head with 4 fluorescent and 3 auto-fluorescent labels) - Supplemental Material See Usage Policy. 5Mb
	Video (MPEG) (Video 4: Truecolor rendering of raw data whole zebrafish with 4 fluorescent and 3 auto-fluorescent labels) - Supplemental Material See Usage Policy. 4Mb
	Video (MPEG) (Video 5: Rendering of HySP processed 4D zebrafish head) - Supplemental Material See Usage Policy. 7Mb
	Video (MPEG) (Video 6: Rendering of HySP processed 4D whole zebrafish) - Supplemental Material See Usage Policy. 7Mb
	Video (MPEG) (Video 7: Rendering of HySP processed 5D zebrafish volumetric timelapse) - Supplemental Material See Usage Policy. 4Mb
	Video (MPEG) (Video 8: Rendering of HySP processed 5D zebrafish volumetric timelapse with 2x1 tiling) - Supplemental Material See Usage Policy. 8Mb
	Video (MPEG) (Video 9: Rendering of HySP processed 5D zebrafish volumetric timelapse with 3x1 tiling) - Supplemental Material See Usage Policy. 6Mb
	Video (MPEG) (Video 10: Overlapped Rendering of HySP processed 5D zebrafish volumetric timelapse) - Supplemental Material See Usage Policy. 3671Kb
	Video (MPEG) (Video 11: Rendering result of multiple unmixing strategies) - Supplemental Material See Usage Policy. 25Mb
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Abstract

Time-lapse imaging of multiple labels is challenging for biological imaging as noise, photobleaching and phototoxicity compromise signal quality, while throughput can be limited by processing time. Here, we report software called Hyper-Spectral Phasors (HySP) for denoising and unmixing multiple spectrally overlapping fluorophores in a low signal-to-noise regime with fast analysis. We show that HySP enables unmixing of seven signals in time-lapse imaging of living zebrafish embryos.

Item Type:

Article

Related URLs:

URL	URL Type	Description
http://dx.doi.org/10.1038/nmeth.4134	DOI	Article
http://www.nature.com/nmeth/journal/v14/n2/full/nmeth.4134.html	Publisher	Article
http://readcube.com/view/10.1038/nmeth.4134	Publisher	Free ReadCube access

ORCID:

Author	ORCID
Trivedi, Vikas	0000-0003-0953-0553
Fraser, Scott E.	0000-0002-5377-0223

Additional Information:

© 2017 Macmillan Publishers Limited, part of Springer Nature. Received 18 July 2016 Accepted 08 November 2016 Published online 09 January 2017. The authors thank T.V. Truong, C. Arnesano, M. Kitano, S. Restrepo (Translational Imaging Center, University of Southern California) and G.H. Bearman for helpful discussions. This work was supported by grants from the Moore Foundation, the Coulter Foundation and the NIH (R01 HD075605, R01 OD019037). We thank C. Paquette for fish care. Author Contributions: F.C. and V.T. performed experiments and analyzed the results. J.M.C., L.A.T. and S.E.F. helped in the experimental design and data analysis. V.T. derived equations. F.C. wrote the software. M.S.A. prepared the samples. V.T., F.C., L.A.T., C.-L.C. and S.E.F. wrote the manuscript. Competing Financial Interests: The authors declare competing financial interests: details are available in the online version of the paper.

Funders:

Funding Agency	Grant Number
Gordon and Betty Moore Foundation	UNSPECIFIED
Coulter Foundation	UNSPECIFIED
NIH	R01 HD075605
NIH	R01 OD019037

Subject Keywords:

Developmental biology; Fluorescence imaging; Image processing

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Record Number:

CaltechAUTHORS:20170117-091624181

Persistent URL:

https://resolver.caltech.edu/CaltechAUTHORS:20170117-091624181

Official Citation:

Hyperspectral phasor analysis enables multiplexed 5D in vivo imaging Francesco Cutrale, Vikas Trivedi, Le A Trinh, Chi-Li Chiu, John M Choi, Marcela S Artiga & Scott E Fraser Nature Methods 14, 149–152 (2017) doi:10.1038/nmeth.4134

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ID Code:

73517

Collection:

CaltechAUTHORS

Deposited By:

Ruth Sustaita

Deposited On:

17 Jan 2017 19:11

Last Modified:

03 Oct 2019 16:28

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