A novel microscopy platform with enhanced detection sensitivity could allow for more comprehensive, system-wide labeling and imaging of greater numbers of biomolecules in living cells and tissues than is currently possible. The platform, called electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy, offers high levels of sensitivity and selectivity. The study, performed by a research team at Columbia University, also details the creation of new molecules that, when paired with the team’s novel microscopy method, allow for the simultaneous labeling and imaging of up to 24 specific biomolecules. The researchers believe this to be nearly five times the number of biomolecules that can be imaged at the same time using existing microscopy techniques. Researchers at Columbia University developed a new optical microscopy platform called electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy that combines a high level of sensitivity and selectivity. Courtesy of Nicoletta Barolini, Columbia University. Researchers used stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). They created a palette of triple-bond-conjugated NIR dyes, each of which displayed a single peak in the cell-silent Raman spectral window. When combined with available fluorescent probes, this palette was found to provide 24 resolvable colors. The amplified “color palette” of molecules broadens the tagging capabilities of the platform, allowing for the imaging of up to 24 structures at a time instead of being limited by only five fluorescent colors, as in fluorescence microscopy. The researchers believe there is potential to further expand the number of structures that can be imaged at one time. The team tested the epr-SRS platform in brain tissue. Proof-of-principle experiments revealed cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-color optical imaging approach for elucidating intricate interactions in complex biological systems. “We were able to see the different cells working together,” said professor Wei Min. “That’s the power of a larger color palette. We can now light up all these different structures in brain tissue simultaneously. In the future we hope to watch them function in real time.” Brain tissue is not the only type of live tissue the researchers envision this technique being used for, Lin added. “Different cell types have different functions, and scientists usually study only one cell type at a time. With more colors, we can now start to study multiple cells simultaneously to observe how they interact and function both on their own and together in healthy conditions versus in disease states.” The team believes that its platform, a hybrid of existing microscopy techniques, combines some of the best features of fluorescence and Raman microscopy. Conventional fluorescence microscopy, while extremely sensitive, limits researchers to seeing a maximum of five structures at a time. Traditional Raman spectroscopy produces more highly defined colors but is less sensitive than fluorescence microscopy. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. The technique allows for the imaging of up to 24 biomolecular structures at a time instead of being limited by only five fluorescent proteins. Courtesy of Nicoletta Barolini, Columbia University. “In the era of systems biology, how to simultaneously image a large number of molecular species inside cells with high sensitivity and specificity remains a grand challenge of optical microscopy,” said Min. “What makes our work new and unique is that there are two synergistic pieces — instrumentation and molecules — working together to combat this long-standing obstacle. Our platform has the capacity to transform understanding of complex biological systems: the vast human cell map, metabolic pathways, the functions of various structures within the brain, the internal environment of tumors, and macromolecule assembly, to name just a few.” The technique could one day be used to treat tumors that are difficult to kill using available drug therapies. “If we can see how structures are interacting in cancer cells, we can identify ways to target specific structures more precisely,” Min said. “This platform could be game-changing in the pursuit of understanding anything that has a lot of components.” The research was published in Nature (doi:10.1038/nature22051).