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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes is a new platform for highly multiplexed epitope-based protein imaging. Here, we present a practical guide including antibody preparation, tissue sample staining, SRS microscope assembly, and epr-SRS tissue imaging.

Abstract

Visualizing a vast scope of specific biomarkers in tissues plays a vital role in exploring the intricate organizations of complex biological systems. Hence, highly multiplexed imaging technologies have been increasingly appreciated. Here, we describe an emerging platform of highly-multiplexed vibrational imaging of specific proteins with comparable sensitivity to standard immunofluorescence via electronic pre-resonance stimulated Raman scattering (epr-SRS) imaging of rainbow-like Raman dyes. This method circumvents the limit of spectrally-resolvable channels in conventional immunofluorescence and provides a one-shot optical approach to interrogate multiple markers in tissues with subcellular resolution. It is generally compatible with standard tissue preparations, including paraformaldehyde-fixed tissues, frozen tissues, and formalin-fixed paraffin-embedded (FFPE) human tissues. We envisage this platform will provide a more comprehensive picture of protein interactions of biological specimens, particularly for thick intact tissues. This protocol provides the workflow from antibody preparation to tissue sample staining, to SRS microscope assembly, to epr-SRS tissue imaging.

Introduction

Complex tissue systems are composed of distinct cellular subpopulations whose spatial locations and interaction networks are deeply intertwined with their functions and dysfunctions1,2. To reveal the tissue architecture and interrogate its complexity, knowledge of the spatial locations of proteins at single-cell resolution is essential. Hence, highly multiplexed protein-imaging technologies have been increasingly appreciated and could become a cornerstone for studying tissue biology3,4,5. Current common multiplexed pr....

Protocol

The protocol was conducted in accordance with the animal experimental protocol (AC-AABD1552) approved by the Institutional Animal Care and Use Committee at Columbia University.

1. Preparation of Raman-dye-conjugated antibodies

  1. Prepare the conjugation buffer as ~0.1 M NaHCO3 in PBS buffer, pH = 8.3, store at 4 °C.
  2. Prepare N-hydroxysuccinimidy (NHS) ester-functionated MARS probe (Supplementary Material) solution as 3 mM in a.......

Representative Results

Figure 3 shows example images of epr-SRS in different samples, including fixed cells (Figure 3A), paraformaldehyde (PFA)-fixed mouse tissues (Figure 3B), and formalin-fixed paraffin-embedded (FFPE) human specimens (Figure 3C). The spatial resolution of SRS microscopy is diffraction-limited, the typical lateral resolution is ~300 nm, and the axial resolution is 1-2 µm using near-infrared light .......

Discussion

Here, we present the immuno-eprSRS protocol which is broadly applicable to common tissue types, including freshly-preserved mouse tissues, FFPE human tissues, and frozen mouse tissues. Immuno-eprSRS has been validated for a panel of epitopes in cells and tissues, as listed in Table 1. This one-shot platform is particularly suitable for applications where cyclic strategies do not function well. For example, cyclic fluorescence is demanding for thick tissues as multiple rounds of 3D immunolabeling are unpr.......

Acknowledgements

We thank Ruth A. Singer and Richard K.P. Benninger for providing mouse pancreas tissues. W.M. acknowledges support from NIH R01 (GM128214), R01 (GM132860), R01 (EB029523) and US Army (W911NF-19-1-0214).

....

Materials

NameCompanyCatalog NumberComments
16% Paraformaldehyde, EM GradeElectron Microscopy Sciences15710
α-tubulinAbcamab18251Primary antibodies
α-tubulinBioLegend625902Primary antibodies
β-III-tubulinBioLegend657402Primary antibodies
β-III-tubulinAbcamab41489Primary antibodies
β-tubulinAbcamab131205Primary antibodies
Agarose, low gellling temperatureSigma AldrichA9414For brain embedding
Anti-a-tubulin antibody produced in rabbit (α-tubulin)Abcamab52866Primary antibodies
Anti-Calbindin antibody produced in mouse (Calbindin)Abcamab82812Primary antibodies
Anti-GABA B receptor R2  antibody produced in guinea pig (GABA B receptor R2)Millipore SigmaAB2255Primary antibodies
Anti-GFAP antibody produced in goat (GFAP)Thermo ScientificPA5-18598Primary antibodies
Anti-Glucagon  antibody produced in mouse (Glucagon)Santa Cruz Biotechnologysc-514592Primary antibodies
Anti-insulin antibody produced in guinea pig (insulin)DAKOIR00261-2Primary antibodies
Anti-MBP antibody produced in rat (MBP)Abcamab7349Primary antibodies
Anti-NeuN antibody produced in rabbit (NeuN)Thermo ScientificPA5-78639Primary antibodies
Anti-Pancreatic polypeptide (PP) antibody produced in goat- Pancreatic polypeptide (PP)Sigma AldrichSAB2500747Primary antibodies
Anti-Pdx1 antibody produced in rabbit (Pdx1)Milipore06-1379Primary antibodies
Anti-Somatostatin antibody produced in rat (Somatostatin)Abcamab30788Primary antibodies
Anti-Vimentin antibody produced in chicken (Vimentin)Abcamab24525Primary antibodies
Band-pass filterKR ElectronicsKR27248 MHz
BNC 50 Ohm TerminatorMini CircuitsSTRM-50
BNC cableThorlabs2249-CCoaxial Cable, BNC Male / Male
Broadband dielectric mirrorThorlabsBB1-E03750 - 1100 nm
C57BL/6J miceJackson Laboratory000664
Centrifuge
CondenserOlympusoil immersion, 1.4 N.A.
Cytokeratin 18Abcamab7797Primary antibodies
Cytokeratin 18Abcamab24561Primary antibodies
DC power supplyTopWard6302DBias voltage is 64 V
Dichroic mountThorlabsKM100CLKinematic Mount for up to 1.3" (33 mm) Tall Rectangular Optics, Left Handed
Donkey anti-Chicken IgY (H+L)Jackson ImmunoResearch703-005-155Secondary antibodies for MARS conjugation
Donkey anti-Goat IgG (H+L)Jackson ImmunoResearch705-005-147Secondary antibodies for MARS conjugation
Donkey anti-Guinea Pig IgG (H+L)Jackson ImmunoResearch706-005-148Secondary antibodies for MARS conjugation
Donkey anti-Mouse IgG (H+L)Jackson ImmunoResearch715-005-151Secondary antibodies for MARS conjugation
Donkey anti-Rabbit IgG (H+L)Jackson ImmunoResearch711-005-152Secondary antibodies for MARS conjugation
Donkey anti-Rat IgG (H+L)Jackson ImmunoResearch712-005-153Secondary antibodies for MARS conjugation
Donkey anti-Sheep IgG (H+L)Jackson ImmunoResearch713-005-147Secondary antibodies for MARS conjugation
DPBSFisher Scientific14-190-250
EpCAMAbcamab71916Primary antibodies
EthanolSigma Aldrich443611
Fast-speed look-in amplifierZurich InstrumentsHF2LIDC - 50 MHz
FFPE Kidney SampleUSBiomaxHuFPT072
FibrillarinAbcamab5821Primary antibodies
GiantinAbcamab24586Primary antibodies
GlucagonSanta Cruz Biotechnologysc-514592Primary antibodies
H2BAbcamab1790Primary antibodies
HeLaATCCATCC CCL-2
High O.D. bandpass filterChroma TechnologyET890/220mFilter the Stokes beam and transmit the pump beam
Hydrophobic penFisher ScientificNC1384846
InsulinThermoFisher701265Primary antibodies
Integrated SRS laser systemApplied Physics & Electronics, Inc.picoEMERALDpicoEMERALD provides an output pulse train at 1,064 nm with 6-ps pulse width and 80-MHz repetition rate, which serves as the Stokes beam. The frequency doubled beam at 532 nm is used to synchronously seed a picosecond optical parametric oscillator (OPO) to produce a mode-locked pulse train with five~6 ps pulse width (the idler beam of the OPO is blocked with an interferometric filter). The output wavelength of the OPO is tunable from 720–950 nm, which serves as the pump beam. The intensity of the 1,064-nm Stokes beam is modulated sinusoidally by a built-in EOM at 8 MHz with a modulation depth of more than 90%. The pump beam is spatially overlapped with the Stokes beam by using a dichroic mirror inside picoEMERALD. The temporal overlap between pump and Stokes pulse trains is achieved with a built-in delay stage and optimized by the SRS signal of pure D2O at the microscope.
Inverted laser-scanning microscopeOlympusFV1200MPE
Kinematic mirror mountThorlabsPOLARIS-K1-2AH2 Low-Profile Hex Adjusters
Lectin from Triticum vulgaris (wheat)Sigma AldrichL0636-5 mg
Long-pass dichroic beam splitterSemrockDi02-R980-25x36980 nm laser BrightLine single-edge laser-flat dichroic beamsplitter
MAP2BioLegend801810Primary antibodies
Microscopy imaging softwareOlympusFluoView
NanoQuant PlateTecanFor absorbance-based, small volume analyses in a plate reader.
Normal donkey serumJackson ImmunoResearch017-000-121
NucBlue Fixed Cell ReadyProbes Reagent (DAPI)Thermo ScientificR37606
Nunc 4-Well DishesFisher Scientific12-566-300
Objective lensOlympusXLPlan Nx25, 1.05-NA, MP, working distance = 2 mm
Paint brush
Periscope assemblyThorlabsRS99includes the top and bottom units, Ø1" post, and clamping fork.
pH meter
Plate readerTecanInfinite 200 PROAn easy-to-use multimode plate reader. Absorbance measurement capabilities over a spectral range of 230–1000 nm.
ProLong Gold antifade reagentThermo ScientificP36930
PSD95Invitrogen51-6900Primary antibodies
Sephadex G-25 MediumGE Life Sciences17-0033-01gel filtration resin for desalting and buffer exchange
Shielded box with BNC connectorsPomona Electronics2902Aluminum Box With Cover, BNC Female/Female
Si photodiodeThorlabsFDS1010350–1100 nm, 10 mm x 10 mm Active Area
Synapsin 2ThermoFisherOSS00073GPrimary antibodies
Tissue Path Superfrost Plus Gold SlidesFisher Scientific22-035813Adhesive slide to attract and chemically bond fresh or formalin-fixed tissue sections firmly to the slide surface (tiisue bindling glass slides)
Triton X-100Fisher ScientificBP151-500
VibratomeLeicaVT1000
VimentinAbcamab8069Primary antibodies
XylenesSigma Aldrich214736

References

  1. Goltsev, Y., et al. Deep profiling of mouse splenic architecture with CODEX multiplexed imaging. Cell. 174 (4), 968-981 (2018).
  2. Taube, J. M., et al.

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