Stimulated raman spectroscopy based multiplexed virtual immunohistology using alkynic, nitrile, or azide probes

A system for examining a tissue sample using stimulated Raman spectroscopy, comprising:

• a pump laser configured to produce a first beam of light at a first wavelength;
• a Stokes beam source configured to produce a second beam of light at at least a second wavelength, the second wavelength different from the first wavelength;
• a plurality of optical elements;
• at least one photodetector configured to detect Raman scattering light and produce signals representative of the detected Raman scattering light;
• and
• a control unit in communication with pump laser, the Stokes beam source, the at least one photodetector, the plurality of optical elements, and a non-transitory memory storing instructions, which instructions when executed cause the processor to:
• control the pump laser, the Stokes beam source, and at least one of the plurality of optical elements to produce a combined output using the first beam of light and the second beam of light;
• cause a tissue sample prepared with at least one target molecule having a targeting agent conjugated with a Raman silent dye (RSD), the targeting agent configured to bind with at least one biomarker, to be interrogated with the combined output and produce Raman scattering light as a result of the interrogation;
• control the at least one photodetector to detect at least a portion of the Raman scattering light and produce signals representative of the detected Raman scattering light;
• and
• produce immunohistological data relating to the tissue sample using the signals representative of the detected Raman scattering light.

A method of examining a tissue sample using stimulated Raman spectroscopy, comprising:

• preparing a tissue sample with at least one target molecule having a targeting agent conjugated with a Raman silent dye (RSD), the targeting agent configured to bind with at least one biomarker;
• producing a first beam of light at a first wavelength using a pump laser;
• producing a second beam of light at at least a second wavelength, the second wavelength different from the first wavelength;
• combining the first beam of light and the second beam of light to provide a combined output;
• interrogating the prepared tissue sample with the combined output to produce Raman scattering light;
• detecting at least a portion of the produced Raman scattering light using at least one photodetector, the photodetector producing signals representative of the detected Raman scattering light;
• and
• producing immunohistological data relating to the tissue sample using the signals representative of the detected Raman scattering light.