Researchers at Columbia University have made a significant step toward breaking the so-called “color barrier” of light microscopy for biological systems, allowing for much more comprehensive, system-wide labeling and imaging of a greater number of biomolecules in living cells and tissues than is currently attainable. The advancement has the potential for many future applications, including helping to guide the development of therapies to treat and cure disease.
“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 Associate Professor of Chemistry Wei 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.”
All existing methods of observing a variety of structures in living cells and tissues have their own strengths, but all are also hindered by fundamental limitations, not the least of which is the existence of a “color barrier.” Fluorescence microscopy, for example, is extremely sensitive and, as such, is the most prevalent technique used in biology labs. The microscope allows scientists to monitor cellular processes in living systems by using proteins that are broadly referred to as “fluorescent proteins” with usually up to five colors. Each of the fluorescent proteins has a target structure to which it applies a “tag,” or color. The five fluorescent proteins, or colors, typically used to tag these structures are BFP (Blue Fluorescent Protein), ECFP (Cyan Fluorescent Protein), GFP (Green Fluorescent Protein), mVenus (Yellow Fluorescent Protein), and DsRed (Red Fluorescent Protein). However, this process imposes the limitation of only being able to view five structures at a time on a single tissue sample.
In addition to fluorescence microscopy, there are a variety of Raman microscopy techniques for observing living cell and tissue structures that work by making visible the vibrations stemming from characteristic chemical bonds in structures. Traditional Raman microscopy produces the highly-defined colors lacking in fluorescence microscopy, but is missing the sensitivity. It therefore requires a strong, concentrated vibrational signal that can only be achieved through the presence of millions of structures with the same chemical bond. If the signal from the chemical bonds is not strong enough, visualizing the associated structure is nearly impossible.
To address this challenge, Min and his team, including Professors Virginia Cornish, chemistry and Rafael Yuste, neuroscience, pursued a novel hybrid of existing microscopy techniques. They developed a new platform called electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy, which combines the best of both worlds, bringing together high levels of sensitivity and selectivity. The technique identifies, with extreme specificity, structures with significantly lower concentrations - instead of millions of the same structure needed to identify the presence of that structure in traditional Raman microscopy, the new instrument requires only 30 for identification. The technique also utilizes a novel set of tagging molecules designed by the team to work synergistically with the new technology. The amplified “color palette” of molecules broadens tagging capabilities, allowing for the imaging of up to 24 structures at a time instead of being limited by only five fluorescent colors. The researchers believe there's potential for even further expansion in the future.
The team has successfully tested the epr-SRS platform in brain tissue. “We were able to see the different cells working together,” Wei said. “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 thing the researchers envision this technique being used for, she 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 new platform has many potential applications, Min said, adding that it is possible the technique could one day be used in the treatment of tumors that are hard to kill with available drugs. “If we can see how structures are interacting in cancer cells, we can identify ways to target specific structures more precisely,” he said. “This platform could be game-changing in the pursuit of understanding anything that has a lot of components.”