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Expanding the Range of Bioorthogonal Tags for Multiplex Stimulated Raman Scattering Microscopy |
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| Authors: | Neville Murphy Dr. William J. Tipping Henry J. Braddick Dr. Liam T. Wilson Prof. Nicholas C. O. Tomkinson Prof. Karen Faulds Prof. Duncan Graham Dr. Pau Farràs |
| Affiliation: | 1. School of Biological and Chemical Sciences, University of Galway, Galway, H91CF50 Ireland CÚRAM, The SFI Research Centre for Medical Devices, University of Galway, Galway, H91 W2TY Ireland These authors contributed equally to this work.;2. Centre for Molecular Nanometrology, WestCHEM, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, G1 1RD United Kingdom;3. Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, G1 1XL United Kingdom;4. School of Biological and Chemical Sciences, University of Galway, Galway, H91CF50 Ireland |
| Abstract: | 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. |
| Keywords: | Bioorthogonal Labeling Imaging Probes Raman Microscopy Spectral Phasor Analysis Stimulated Raman Scattering |