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Nw: Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing

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Abstract

Mapping the localization of a pair of proteins in their native three-dimensional (3D) context would possibly be precious across many areas of biomedicine, but multiplexed fluorescence imaging has restricted intrinsic multiplexing skill, and most techniques for increasing multiplexity can easiest be utilized to skinny samples (

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    AcknowledgementsWe thank R. Yuste, R. Tomer, L. Wei, F. Hu and C. Chen for useful d iscussions. WM acknowledges give a enhance to from NIH R01 (GM128214), R01 (GM132860), R01 (EB029523) and US Military (W911NF-19-1-0214).

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    Prolonged Data Fig. 1 Testing spectral compatibility between Raman-inviting dyes and tissue clearing techniques.

    a

    , b

    , Fluorescence ( a

    ) and epr-SRS imaging at 1604 cm

    , epr-SRS imaging at 1642 cm −1 of ATTO 740 stained GFAP on PFA-mounted mouse mind tissue inner different R IMS.

    )d

    , Raman spectra of PBS and RIMS easiest at 1500–1700 cm

    −1 (spectral window for vibrational pe aks of most industrial dyes). Some spectra were amplified by lumber folds (quantity inner) to area them under an identical scale.

    )e

    , epr-SRS imaging at 2147 cm −1 of MARS2147 stained GFAP on PFA-mounted mouse mind tissue inne r different RIMS.

    )f

    , Burly Raman spectra of tissue clearing reagents. The pink area signifies the interested spectral window (2000–2400 cm

    −1), by which the nitrile-bond newshounds in MARS probes vibrate. Spectra in (

    α-tubulin in COS7 cells were stained with industrial dye ATTO 740 (
    a-b) and MARS2228 ( cd ).

    )b

    , d

    , Magnified regions outlined by the inexperienced box in ( a, c), respectively. Under, epr-SRS worth alongside the white dotted line cleave in the magnified photographs; curves were drawn with a spline fit. Scale bars, 20 µm in (

    a, c); 5 µm in ( b, d

    ).

    Prolonged Data Fig. 3 Immuno-eprSRS imaging with MARS probes in tissue samples.

    a, More than one protein targets are like minded with immuno-eprSRS with C-cored MARS probes (in blue ) and O-cored MARS probes (in red). Targeted proteins were stained on PFA-mounted mouse mind or cerebellum sections (40-μm thick) with oblique immunostaining.

    )b

    , Mosaic imaging with motorized sample stage. NeuN, neuronal nuclei; TUBB3, β-III-tubulin; CB, calbindin; GABBR2, GABA B receptor 2; PSD95, postsynaptic density protein 95; MAP2, microtubule linked protein 2; GFAP, glial fibrillary acidic protein; MBP, myelin general protein.

    )c

    , Teach immunostaining with MARS-probe conjugated main antibodies.

    )d

    , MARS probe-conjugated lectin staining on PFA-mounted cerebellum tissue sections. WGA, wheat germ agglutinin; LEL, Lycopersicon Esculentum lectin. More antibody files is in Supplementary Desk 3

    . Scale bars, 50 µm in (

    a) and ( cd ); 200 µm in ( b).

    Prolonged Data Fig . 4 Quantitative comparison of the pricetag-to-noise ratios of immuno-eprSRS with long-established immunofluorescence.

    a, Immunostained GFAP in 40-µm thick mouse mind tissue.

    )b

    , Immunostained NeuN in 40-µm thick mouse mind tissue. Every files level of the pricetag turned into calculated because the averaged fluorescence/epr-SRS worth of particular person nucleus for NeuN or particular person astrocytes for GFAP; the noise turned into measured because the sd from tissue areas with out stained constructions. Pixel dwell time is 2 µs for fluorescence and 80 µs for epr-SRS. Scale bars, 50 µm.

    a, Two-color imaging tests on mounted cells (nucleus protein H2B labeled by O-cored MARS and cytoskeleton protein β-tubulin labeled by C-cored dyes). No obvious spoiled-talk between two channels turned into noticed. (left) β-tubulin (labeled by MARS2176) and H2B (labeled by MARS2188). (factual) β-tubulin (labeled by MARS2176) and H2B (labeled by MARS2242).

    )b

    , A workflow of multiplexed labeling (also see techniques).

    )c

    , d

    , Simultaneous twelve-target protein imaging on cerebellum tissue sections from ( c) postnatal 25 days (P25) and (
    d
    ) postnatal 15 days (P15) mice. Fluorescence: DAPI (entire DNA), vesicular glutamate transporter 1 (VGluT1-Alexa Fluor 488, Glutamatergic neurons, bid immunolabeling), tyrosine hydroxylase (TH-Alexa Fluor 594, Dopaminergic neurons, bid immunolabeling), actin (Phalloidin-Alexa Fluor 647) ; epr-SRS: neuronal nuclei (NeuN, neurons, MARS2228), α-tubulin-MARS2176 (bid immunolabeling), calbindin (CB, Purkinje neurons, MARS2145), β-III-tubulin (TUBB3, neurons, MARS2200), wheat germ agglutinin (WGA-MARS2242), GABA B receptor 2 (GABBR2, GABAergic neurons, MARS2212), myelin general protein (MBP; oligodendrocytes, MARS2188), glial fibrillary acidic protein (GFAP, astrocytes and neural stem cells, MARS2159). Scale bars, 20 µm in (

    a); 50 µm in ( cd

    ).

    Prolonged Data Fig. 6 Sectioning skill and axial resolution of immuno-eprSRS imaging.

    a, Quantity-rendered image;

    )b

    , orthogonal views;

    )c

    , Zoomed-in quantity-rendered image of ( a );

    )d

    , optically zoomed-in quantity-rendered image of MARS2159 stained GFAP in 100-μm thick mouse mind tissue. The inset yellow box in (

    Prolong ed Data Fig. 7 Tests on immuno-eprSRS for volumetric imaging.

    a, Photos of 1-mm thick mind slices with out clearing (uncleared), cleared by 8 M urea and 0.2% Triton for 2 days and cleared by uDISCO. Scale bar, 2 mm.

    )b

    , Normalized absorption spectra of MARS probes in RI matching alternatives in BABB, 3DISCO (DBE) and uDISCO (BABB-D4). A bluer absorption top looks in DBE, but no longer in BABB and BABB-D4.

    Data Fig. 8 Volumetric immuno-eprSRS imaging on 500-μm-thick mouse cerebellum sections with uDISCO clearing.

    a

    , Quantity-rendered image of GFAP (astrocytes) labeled with O-cored MARS2159 in 500-μm thick cerebellum sections cleared by uDISCO.

    )b

    , Quantity-rendered image of NeuN (neuronal nucleus) labeled with C-cored MARS2228 in 500-μm thick cerebellum sections cleared by uDISCO. Enlarged photographs of Fig.

    4a. Single-airplane photographs screen loyal epr-SRS inequity alongside your total depth for every and each samples. Scale bars, 100 µm.

    a

    , Quantity-rendered photographs of GFAP (astrocytes, labeled with MARS2228) and MBP (oligodendrocytes, labeled with Alexa Fluor 488) in 1-mm thick cerebellum sections cleared by uDISCO. Two-color merged single-airplane photographs also screen loyal epr-SRS and fluorescence inequity alongside your total depth. Scale bars, 100 µm.

    )b

    , SRS worth-to-noise ratio enchancment of rDISCO over uDISCO at different depths.

    Prolonged Data Fig. 10 Quantitative 3D analyzes on multiplexed volumetric photographs.

    a, Segmentation of 4 anatomical layers as white topic, the granular layer, the Purkinje layer and the molecular layer. Scale bar, 100 µm.

    )b

    , Correlation heatmaps between randomized photographs. (up) Color bar plotted in the vary of . (down) Color bar plotted in the vary of [−0.02 0.02].

    )c

    , Compositional percentages of Vim

    +/GFAP

    –, Vim

    Supplementary files

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    Shi, L., Wei, M., Miao, Y. et al. Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing. Nat Biotechnol ) (2021). https://doi.org/10.1038/s41587-021-01041-z

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    https://doi.org/10.1038/s41587-021-01041-z

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