A subscription to JoVE is required to view this content. Sign in or start your free trial.
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.
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.
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....
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
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 .......
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.......
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).
....Name | Company | Catalog Number | Comments |
16% Paraformaldehyde, EM Grade | Electron Microscopy Sciences | 15710 | |
α-tubulin | Abcam | ab18251 | Primary antibodies |
α-tubulin | BioLegend | 625902 | Primary antibodies |
β-III-tubulin | BioLegend | 657402 | Primary antibodies |
β-III-tubulin | Abcam | ab41489 | Primary antibodies |
β-tubulin | Abcam | ab131205 | Primary antibodies |
Agarose, low gellling temperature | Sigma Aldrich | A9414 | For brain embedding |
Anti-a-tubulin antibody produced in rabbit (α-tubulin) | Abcam | ab52866 | Primary antibodies |
Anti-Calbindin antibody produced in mouse (Calbindin) | Abcam | ab82812 | Primary antibodies |
Anti-GABA B receptor R2Â antibody produced in guinea pig (GABA B receptor R2) | Millipore Sigma | AB2255 | Primary antibodies |
Anti-GFAP antibody produced in goat (GFAP) | Thermo Scientific | PA5-18598 | Primary antibodies |
Anti-Glucagon antibody produced in mouse (Glucagon) | Santa Cruz Biotechnology | sc-514592 | Primary antibodies |
Anti-insulin antibody produced in guinea pig (insulin) | DAKO | IR00261-2 | Primary antibodies |
Anti-MBP antibody produced in rat (MBP) | Abcam | ab7349 | Primary antibodies |
Anti-NeuN antibody produced in rabbit (NeuN) | Thermo Scientific | PA5-78639 | Primary antibodies |
Anti-Pancreatic polypeptide (PP) antibody produced in goat- Pancreatic polypeptide (PP) | Sigma Aldrich | SAB2500747 | Primary antibodies |
Anti-Pdx1 antibody produced in rabbit (Pdx1) | Milipore | 06-1379 | Primary antibodies |
Anti-Somatostatin antibody produced in rat (Somatostatin) | Abcam | ab30788 | Primary antibodies |
Anti-Vimentin antibody produced in chicken (Vimentin) | Abcam | ab24525 | Primary antibodies |
Band-pass filter | KR Electronics | KR2724 | 8 MHz |
BNC 50 Ohm Terminator | Mini Circuits | STRM-50 | |
BNC cable | Thorlabs | 2249-C | Coaxial Cable, BNC Male / Male |
Broadband dielectric mirror | Thorlabs | BB1-E03 | 750 - 1100 nm |
C57BL/6J mice | Jackson Laboratory | 000664 | |
Centrifuge | |||
Condenser | Olympus | oil immersion, 1.4 N.A. | |
Cytokeratin 18 | Abcam | ab7797 | Primary antibodies |
Cytokeratin 18 | Abcam | ab24561 | Primary antibodies |
DC power supply | TopWard | 6302D | Bias voltage is 64 V |
Dichroic mount | Thorlabs | KM100CL | Kinematic Mount for up to 1.3" (33 mm) Tall Rectangular Optics, Left Handed |
Donkey anti-Chicken IgY (H+L) | Jackson ImmunoResearch | 703-005-155 | Secondary antibodies for MARS conjugation |
Donkey anti-Goat IgG (H+L) | Jackson ImmunoResearch | 705-005-147 | Secondary antibodies for MARS conjugation |
Donkey anti-Guinea Pig IgG (H+L) | Jackson ImmunoResearch | 706-005-148 | Secondary antibodies for MARS conjugation |
Donkey anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 715-005-151 | Secondary antibodies for MARS conjugation |
Donkey anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-005-152 | Secondary antibodies for MARS conjugation |
Donkey anti-Rat IgG (H+L) | Jackson ImmunoResearch | 712-005-153 | Secondary antibodies for MARS conjugation |
Donkey anti-Sheep IgG (H+L) | Jackson ImmunoResearch | 713-005-147 | Secondary antibodies for MARS conjugation |
DPBS | Fisher Scientific | 14-190-250 | |
EpCAM | Abcam | ab71916 | Primary antibodies |
Ethanol | Sigma Aldrich | 443611 | |
Fast-speed look-in amplifier | Zurich Instruments | HF2LI | DC - 50 MHz |
FFPE Kidney Sample | USBiomax | HuFPT072 | |
Fibrillarin | Abcam | ab5821 | Primary antibodies |
Giantin | Abcam | ab24586 | Primary antibodies |
Glucagon | Santa Cruz Biotechnology | sc-514592 | Primary antibodies |
H2B | Abcam | ab1790 | Primary antibodies |
HeLa | ATCC | ATCC CCL-2 | |
High O.D. bandpass filter | Chroma Technology | ET890/220m | Filter the Stokes beam and transmit the pump beam |
Hydrophobic pen | Fisher Scientific | NC1384846 | |
Insulin | ThermoFisher | 701265 | Primary antibodies |
Integrated SRS laser system | Applied Physics & Electronics, Inc. | picoEMERALD | picoEMERALD 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 microscope | Olympus | FV1200MPE | |
Kinematic mirror mount | Thorlabs | POLARIS-K1-2AH | 2 Low-Profile Hex Adjusters |
Lectin from Triticum vulgaris (wheat) | Sigma Aldrich | L0636-5 mg | |
Long-pass dichroic beam splitter | Semrock | Di02-R980-25x36 | 980 nm laser BrightLine single-edge laser-flat dichroic beamsplitter |
MAP2 | BioLegend | 801810 | Primary antibodies |
Microscopy imaging software | Olympus | FluoView | |
NanoQuant Plate | Tecan | For absorbance-based, small volume analyses in a plate reader. | |
Normal donkey serum | Jackson ImmunoResearch | 017-000-121 | |
NucBlue Fixed Cell ReadyProbes Reagent (DAPI) | Thermo Scientific | R37606 | |
Nunc 4-Well Dishes | Fisher Scientific | 12-566-300 | |
Objective lens | Olympus | XLPlan N | x25, 1.05-NA, MP, working distance = 2 mm |
Paint brush | |||
Periscope assembly | Thorlabs | RS99 | includes the top and bottom units, Ø1" post, and clamping fork. |
pH meter | |||
Plate reader | Tecan | Infinite 200 PRO | An easy-to-use multimode plate reader. Absorbance measurement capabilities over a spectral range of 230–1000 nm. |
ProLong Gold antifade reagent | Thermo Scientific | P36930 | |
PSD95 | Invitrogen | 51-6900 | Primary antibodies |
Sephadex G-25 Medium | GE Life Sciences | 17-0033-01 | gel filtration resin for desalting and buffer exchange |
Shielded box with BNC connectors | Pomona Electronics | 2902 | Aluminum Box With Cover, BNC Female/Female |
Si photodiode | Thorlabs | FDS1010 | 350–1100 nm, 10 mm x 10 mm Active Area |
Synapsin 2 | ThermoFisher | OSS00073G | Primary antibodies |
Tissue Path Superfrost Plus Gold Slides | Fisher Scientific | 22-035813 | Adhesive slide to attract and chemically bond fresh or formalin-fixed tissue sections firmly to the slide surface (tiisue bindling glass slides) |
Triton X-100 | Fisher Scientific | BP151-500 | |
Vibratome | Leica | VT1000 | |
Vimentin | Abcam | ab8069 | Primary antibodies |
Xylenes | Sigma Aldrich | 214736 |
Explore More Articles
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved