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

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 bio‐orthogonal 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.

Biomedical applications, perspectives and tag design concepts in the cell – silent Raman window

“…have demonstrated the potential of metallacarboranes as Raman reporters by employing boron clusters together with a bis-alkyne and deuterated fatty acid to achieve multiplex Raman imaging in live HeLa cells. 64 Activatable Raman probes, a group of molecules able to alternate between “on” and “off” states, have found a unique place in multiplex imaging applications over the past few years. Du and Wei were the first to identify the first general design of photoactivatable alkyne-based Raman probes (Fig.…”