Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy

• Published in: Angewandte Chemie International Edition 2023-11, Vol.62 (48), p.e202311530-n/a
• Main Authors: Murphy, Neville, Tipping, William J., Braddick, Henry J., Wilson, Liam T., Tomkinson, Nicholas C. O., Faulds, Karen, Graham, Duncan, Farràs, Pau
• Format: Article
• Language: English
• Subjects: Alkynes; Bioorthogonal Labeling; Communication; Communications; Deuteration; Fatty Acids; Imaging Probes; Microscopy; Multiplexing; Nonlinear Optical Microscopy - methods; Phasors; Raman Microscopy; Raman spectra; Raman spectroscopy; Spectral Phasor Analysis; Spectrum Analysis, Raman - methods; Stimulated Raman Scattering; Wavelengths
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.202311530

Multiplex optical detection in live cells is challenging due to overlapping signals and poor signal‐to‐noise associated with some chemical reporters. To address this, the application of spectral phasor analysis to stimulated Raman scattering (SRS) microscopy for unmixing three bioorthogonal Raman probes within cells is reported. Triplex detection of a metallacarborane using the B−H stretch at 2480–2650 cm−1, together with a bis‐alkyne and deuterated fatty acid can be achieved within the cell‐silent region of the Raman spectrum. When coupled to imaging in the high‐wavenumber region of the cellular Raman spectrum, nine discrete regions of interest can be spectrally unmixed from the hyperspectral SRS dataset, demonstrating a new capability in the toolkit of multiplexed Raman imaging of live cells. Hyperspectral stimulated Raman scattering (SRS) microscopy with spectral phasor analysis enables the multiplex detection of bioorthogonal Raman groups in cells. Unmixing of the overlapping alkyne and deuterium signals together with the detection of metallacarboranes is reported within the cell‐silent region of the Raman spectrum.