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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

[img] Image (JPEG) (Supplementary Figure 1: Comparison of HySP and linear unmixing under different signal-to-noise ratios (SNRs)) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 2: Errors on hyper-spectral phasor plot) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 3: Sensitivity of phasor point) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 4: Effect of phasor space denoising on scatter error and shifted-mean error) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 5: Effect of phasor space denoising on image intensity) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 6: Phasor analysis for unmixing hyper-spectral fluorescent signals in vivo) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 7: Autofluorescence identification and removal in phasor space) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 8: Optical separation of eGFP and citrine) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 9: Comparison of HySP and linear unmixing in resolving 7 fluorescent signals) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 10: Effect of binning on HySP analysis of 7 in vivo fluorescent signals) - Supplemental Material
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[img] Video (MPEG) (Video 1: Truecolor rendering of raw data zebrafish with 2 fluorescent and 1 auto-fluorescent labels) - Supplemental Material
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[img] Video (MPEG) (Video 2: HySP resolved 2-color zebrafish with autofluorescence removal) - Supplemental Material
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[img] Video (MPEG) (Video 3: Truecolor rendering of raw data zebrafish head with 4 fluorescent and 3 auto-fluorescent labels) - Supplemental Material
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[img] Video (MPEG) (Video 4: Truecolor rendering of raw data whole zebrafish with 4 fluorescent and 3 auto-fluorescent labels) - Supplemental Material
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[img] Video (MPEG) (Video 5: Rendering of HySP processed 4D zebrafish head) - Supplemental Material
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[img] Video (MPEG) (Video 6: Rendering of HySP processed 4D whole zebrafish) - Supplemental Material
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[img] Video (MPEG) (Video 7: Rendering of HySP processed 5D zebrafish volumetric timelapse) - Supplemental Material
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[img] Video (MPEG) (Video 8: Rendering of HySP processed 5D zebrafish volumetric timelapse with 2x1 tiling) - Supplemental Material
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[img] Video (MPEG) (Video 9: Rendering of HySP processed 5D zebrafish volumetric timelapse with 3x1 tiling) - Supplemental Material
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[img] Video (MPEG) (Video 10: Overlapped Rendering of HySP processed 5D zebrafish volumetric timelapse) - Supplemental Material
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[img] Video (MPEG) (Video 11: Rendering result of multiple unmixing strategies) - Supplemental Material
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[img] PDF (Supplementary Figures 1–10, Supplementary Tables 1–3 and Supplementary Notes 1–6) - Supplemental Material
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[img] Archive (ZIP) (Supplementary Software 1) - Supplemental Material
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[img] Archive (ZIP) (Supplementary Software 2) - Supplemental Material
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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:
URLURL TypeDescription
http://dx.doi.org/10.1038/nmeth.4134DOIArticle
http://www.nature.com/nmeth/journal/v14/n2/full/nmeth.4134.htmlPublisherArticle
http://readcube.com/view/10.1038/nmeth.4134PublisherFree ReadCube access
ORCID:
AuthorORCID
Trivedi, Vikas0000-0003-0953-0553
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 AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
Coulter Foundation UNSPECIFIED
NIHR01 HD075605
NIHR01 OD019037
Subject Keywords:Developmental biology; Fluorescence imaging; Image processing
Issue or Number:2
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
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
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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