A new method expands the number of molecules that can be imaged simultaneously in biological samples. Wei Min and coworkers at Columbia University demonstrate that their method could resolve 24 dye-labeled molecules at a time with the method—20 with a new version of stimulated Raman scattering (SRS) microscopy called electronic preresonance SRS and four with fluorescence microscopy (Nature 2017, DOI: 10.1038/nature22051).
The original version of SRS microscopy is a label-free method in which researchers excite a target molecule with a laser wavelength that is far from its absorption wavelength. One downside of the method is that it can’t detect molecules at submillimolar concentrations.
In the new method, the Columbia team attaches light-absorbing Raman-active labels to the target molecules. The researchers excite each label with laser beams tuned just below its absorption frequency. These changes improve the method’s sensitivity to the nanomolar level and enable the simultaneous imaging of multiple biomolecules.
The new probes comprise xanthene scaffolds conjugated to Raman-active nitriles or alkynes. Each probe has a single sharp Raman scattering peak between 1,800 and 2,800 cm−1. Through scaffold modifications and isotope editing, the researchers can tune the vibrational frequency of the Raman peak. They report 14 of the new dyes and show that they can be used with six commercially available Raman labels and four fluorescent dyes. The Raman and fluorescence dyes don’t interfere with one another because they absorb in different spectral regions.
Min and coworkers used up to 16 of these labels to tag and image various molecules in live cells and tissues. They want to use the full palette of probes for complex mixtures such as those in proteomics.
“This is a similar approach to making [surface-enhanced Raman spectroscopy] tags; however, no nanoparticles are necessary and that may reduce biological side-effects,” says Zachary D. Schultz, a spectroscopy expert at the University of Notre Dame. “This is an exciting report and should enable new studies tracking increased numbers of species in biological systems.”