By some estimates, the human eye can distinguish between a million colors, yet scientists looking through a microscope have been limited to seeing only five. Researchers at Columbia University have blasted through this "color barrier" with a new technique that ups the color vision of microscopy to a total of 24 different shades.

Coloring structures inside our cells can help scientists track the effects of drugs, observe how cellular biomechanics play out, trace complex metabolic pathways and more. Till now, there have been two main ways for researchers to use color to observe cellular materials: fluorescence microscopy and Raman spectroscopy.

In fluorescence microscopy, cellular structures are either stained or tagged with a fluorescent chemical known as a fluorophore (or fluorochrome) which, when struck by light, re-emits that light in a particular wavelength, illuminating the structure. However, the system only allows five different colors to be used at once, which means only five different structures can be watched at one time per tissue sample. If researchers want to see other processes in the same tissue, they need to clean it and start again, and the cleaning process can damage the tissue.

With Raman spectroscopy, a tissue sample is beamed with a laser. A part of that light is scattered based on the vibrations it encounters from molecules in the tissue. A Raman microscope translates these different wavelengths into images and provides information about the chemical structure of the tissue. A drawback to this method is that for it to be effective, millions of any one structure must be present for it to create enough of a vibrational frequency to work. Any less and imaging becomes practically impossible.

Raman spectroscopy peers into our tissues by relying on the way in which light is altered by the vibrations of the molecules it encounters(Credit: Nicoletta Barolini, Columbia University)

To solve the issues with these systems, the Columbia researchers combined them both. They call their new technique electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy. Instead of a million, the new microscope only needs 30 structures to perform Raman spectroscopy, making it leagues more sensitive than its precursors. The researchers also harnessed different tagging molecules and stains that allow 24 different cellular structures to be seen instead of just five. Lu Wei, lead author on the study, told New Atlas that 14 of those colors come from tagging molecules created by her team, while six were already are commercially available. The other four come from fluorescent stains.

The epr-SRS system was tested out successfully using brain tissue.

"We were able to see the different cells working together," said Wei. "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."

Not only does the new system allow researchers to get a more comprehensive picture of what's happening inside cells, the enhanced color system lets them understand what's happening between cells as well.

"Different cell types have different functions, and scientists usually study only one cell type at a time," said Wei. "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."

Wei Min, in whose lab the research was conducted, added that the new imaging technique could open the door to better cancer treatments. If researchers can see how structures interact in individual cancer cells, he said, then drugs could be developed to target those structures with increased precision.

"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," Min said. "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 work of the researchers has been published in the journal Nature.

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