Researchers optimize stimulated Raman scattering microscopy and combine it with a new panel of Raman-active dyes to enable 24-color bioimaging.
Fluorescence microscopy has been an invaluable tool for understanding complex biological systems, but it is largely limited to studying a few factors at a time. This limit is determined by the number of colors that can be imaged simultaneously, because fluorophores often have broad, overlapping spectra. Even the most sophisticated methods are limited to imaging around ten fluorophores at once. Methods that can improve multiplexing are needed to help researchers understand complex processes as they occur in cells.
Wei Min at Columbia University and Lu Wei, a postdoctoral fellow in Min's laboratory, sought to develop a multiplexed imaging method that would bypass the barriers imposed by spectral overlap in fluorescence microscopy. For this, they turned to nonlinear Raman imaging: specifically, stimulated Raman scattering (SRS) microscopy. In Raman imaging, spectral peaks are roughly 100 times narrower than fluorescence peaks, which makes colors much easier to distinguish. In SRS microscopy, samples are illuminated with two aligned beams (the pump and Stokes beams). Fine tuning of these beams at multiple wavelengths stimulates molecular vibrations that cause intensity changes in both beams, and these changes can be measured to provide contrast and generate a multicolor image.
Unlike fluorescence microscopy, where fluorescent molecules are excited near their absorption maxima, conventional SRS microscopy uses laser energy levels that are nonresonant, or far below the electronic absorption of molecules. Consequently, conventional SRS is limited to millimolar detection sensitivity. “We were curious what would happen if we brought electronic resonance to the nonlinear Raman imaging,” recalls Min.
This curiosity led them to develop electronic preresonance SRS (epr-SRS), a technique that made it possible to get strong SRS signals from dyes like Atto740 with very low background signal. They found that epr-SRS was around 1,000 times more sensitive than conventional SRS. These results, which show that epr-SRS approaches the sensitivity of confocal fluorescence, surprised Min, although he notes they are consistent with the literature.
Based on these promising findings, the researchers developed epr-SRS microscopy and imaged dye-labeled DNA in fixed mammalian cells. They saw that their method produced images that corresponded well with images obtained by fluorescence microscopy but offered much higher chemical selectivity. They also observed low photobleaching.
They then extended the method for 'super multiplexing'. For this, they developed a set of twenty near-infrared dyes, called the Manhattan Raman scattering (MARS) dyes, based on the xanthene dye scaffold. These dyes were easily separable using epr-SRS microscopy, and they were used for sixteen-color imaging of dye-labeled cells. The researchers also demonstrated eight-color imaging in hippocampal neurons as well as live-cell imaging in mammalian and bacterial cells.
Epr-SRS microscopy can be implemented on commercial SRS and coherent anti-Stokes Raman scattering microscopes, and this should facilitate broad uptake of the method. According to Min, “the future challenge is probably on the labeling side rather than on the imaging side,” as “immunohistochemistry of using a large number of antibodies can sometimes be nontrivial.”
Min also notes that the research was multidisciplinary, requiring scientists with expertise in spectroscopy, microscopy, chemistry and biology to work closely together. He says that future work will involve expanding the palette of available dyes and making them more compatible with labeling living cells. The work as a whole represents a major step forward for highly multiplexed imaging and should encourage more biologists to consider Raman microscopy.
Wei, L., et al. Super-multiplex vibrational imaging. Nature 544, 465–470 (2017).
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Strack, R. Good vibrations for super-multiplexed imaging. Nat Methods 14, 552 (2017). https://doi.org/10.1038/nmeth.4319