ECB-ART-50279
Development
2022 Jul 15;14914:. doi: 10.1242/dev.200545.
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SAIBR: a simple, platform-independent method for spectral autofluorescence correction.
Rodrigues NTL
,
Bland T
,
Borrego-Pinto J
,
Ng K
,
Hirani N
,
Gu Y
,
Foo S
,
Goehring NW
.
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Biological systems are increasingly viewed through a quantitative lens that demands accurate measures of gene expression and local protein concentrations. CRISPR/Cas9 gene tagging has enabled increased use of fluorescence to monitor proteins at or near endogenous levels under native regulatory control. However, owing to typically lower expression levels, experiments using endogenously tagged genes run into limits imposed by autofluorescence (AF). AF is often a particular challenge in wavelengths occupied by commonly used fluorescent proteins (GFP, mNeonGreen). Stimulated by our work in C. elegans, we describe and validate Spectral Autofluorescence Image Correction By Regression (SAIBR), a simple platform-independent protocol and FIJI plug-in to correct for autofluorescence using standard filter sets and illumination conditions. Validated for use in C. elegans embryos, starfish oocytes and fission yeast, SAIBR is ideal for samples with a single dominant AF source; it achieves accurate quantitation of fluorophore signal, and enables reliable detection and quantification of even weakly expressed proteins. Thus, SAIBR provides a highly accessible low-barrier way to incorporate AF correction as standard for researchers working on a broad variety of cell and developmental systems.
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Francis Crick Institute, FC001086 Cancer Research UK, FC001086 Medical Research Council , FC001086 Wellcome Trust , BB/T000481/1 Biotechnology and Biological Sciences Research Council , P40 OD010440 NIH HHS , 220790/Z/20/Z Wellcome Trust
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References [+] :
Alberti,
A single laser method for subtraction of cell autofluorescence in flow cytometry.
1987, Pubmed
Alberti, A single laser method for subtraction of cell autofluorescence in flow cytometry. 1987, Pubmed
An, SKN-1 links C. elegans mesendodermal specification to a conserved oxidative stress response. 2003, Pubmed
Arribere, Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. 2014, Pubmed
Baharlou, AFid: a tool for automated identification and exclusion of autofluorescent objects from microscopy images. 2021, Pubmed
Barkoulas, Robustness and epistasis in the C. elegans vulval signaling network revealed by pathway dosage modulation. 2013, Pubmed
Beatty, The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo. 2010, Pubmed
Billinton, Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. 2001, Pubmed
Borrego-Pinto, Live Imaging of Centriole Dynamics by Fluorescently Tagged Proteins in Starfish Oocyte Meiosis. 2016, Pubmed , Echinobase
Borrego-Pinto, Distinct mechanisms eliminate mother and daughter centrioles in meiosis of starfish oocytes. 2016, Pubmed , Echinobase
Castiglioni, Epidermal PAR-6 and PKC-3 are essential for larval development of C. elegans and organize non-centrosomal microtubules. 2020, Pubmed
Chen, Facile autofluorescence suppression enabling tracking of single viruses in live cells. 2019, Pubmed
Cowen, Pontamine sky blue: a counterstain for background autofluorescence in fluorescence and immunofluorescence histochemistry. 1985, Pubmed
Croce, Autofluorescence spectroscopy and imaging: a tool for biomedical research and diagnosis. 2014, Pubmed
Davis, Accurate detection of low levels of fluorescence emission in autofluorescent background: francisella-infected macrophage cells. 2010, Pubmed
Dickinson, CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering. 2016, Pubmed
Dokshin, Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans. 2018, Pubmed
Gross, Guiding self-organized pattern formation in cell polarity establishment. 2019, Pubmed
Heppert, Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system. 2016, Pubmed
Hermann, Genetic analysis of lysosomal trafficking in Caenorhabditis elegans. 2005, Pubmed
Hoege, LGL can partition the cortex of one-cell Caenorhabditis elegans embryos into two domains. 2010, Pubmed
Kumar, Feasibility of in vivo imaging of fluorescent proteins using lifetime contrast. 2009, Pubmed
Lang, The PAR proteins: from molecular circuits to dynamic self-stabilizing cell polarity. 2017, Pubmed
Laufer, Segregation of developmental potential in early embryos of Caenorhabditis elegans. 1980, Pubmed
Lichten, Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers. 2014, Pubmed
Mansfield, Autofluorescence removal, multiplexing, and automated analysis methods for in-vivo fluorescence imaging. 2005, Pubmed
McRae, Robust blind spectral unmixing for fluorescence microscopy using unsupervised learning. 2019, Pubmed
Neumann, Simple method for reduction of autofluorescence in fluorescence microscopy. 2002, Pubmed
Pang, Autofluorescence removal using a customized filter set. 2013, Pubmed
Pincus, Autofluorescence as a measure of senescence in C. elegans: look to red, not blue or green. 2016, Pubmed
Pintard, Mitotic Cell Division in Caenorhabditis elegans. 2019, Pubmed
Reich, Regulated Activation of the PAR Polarity Network Ensures a Timely and Specific Response to Spatial Cues. 2019, Pubmed
Rodriguez, aPKC Cycles between Functionally Distinct PAR Protein Assemblies to Drive Cell Polarity. 2017, Pubmed
Roederer, Compensation in flow cytometry. 2002, Pubmed
Roederer, Cell-by-cell autofluorescence correction for low signal-to-noise systems: application to epidermal growth factor endocytosis by 3T3 fibroblasts. 1986, Pubmed
Sallee, Apical PAR complex proteins protect against programmed epithelial assaults to create a continuous and functional intestinal lumen. 2021, Pubmed
Shaner, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. 2004, Pubmed
Shcherbo, Far-red fluorescent tags for protein imaging in living tissues. 2009, Pubmed
Shi, In vivo analysis of recycling endosomes in Caenorhabditis elegans. 2015, Pubmed
Steinkamp, Dual-laser, differential fluorescence correction method for reducing cellular background autofluorescence. 1986, Pubmed
Stiernagle, Maintenance of C. elegans. 2006, Pubmed
Szöllösi, Autofluorescence correction for fluorescence in situ hybridization. 1995, Pubmed
Terasaki, Redistribution of cytoplasmic components during germinal vesicle breakdown in starfish oocytes. 1994, Pubmed , Echinobase
Teuscher, Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans. 2018, Pubmed
Tsukamoto, LIN-41 and OMA Ribonucleoprotein Complexes Mediate a Translational Repression-to-Activation Switch Controlling Oocyte Meiotic Maturation and the Oocyte-to-Embryo Transition in Caenorhabditis elegans. 2017, Pubmed
Van de Lest, Elimination of autofluorescence in immunofluorescence microscopy with digital image processing. 1995, Pubmed
Zhang, Tissue morphogenesis: how multiple cells cooperate to generate a tissue. 2010, Pubmed
Zimmermann, Spectral imaging and its applications in live cell microscopy. 2003, Pubmed
Zipperlen, Roles for 147 embryonic lethal genes on C.elegans chromosome I identified by RNA interference and video microscopy. 2001, Pubmed