Innovative Platform for Overcoming Light Microscopy Color Barrier

Scientists from Columbia University have moved one step closer toward breaking the “color barrier” of light microscopy for biological systems, thus enabling more system-wide and comprehensive labeling and imaging of innumerable biomolecules in living cells and tissues than achievable at present.

This progress has the ability to assist in a number of prospective applications, such as guiding the advancement of therapies to treat and cure various diseases.

The research team headed by Associate Professor of Chemistry Wei Min has reported the creation of an innovative optical microscopy platform with dramatically improved detection sensitivity in a study published online in the journal Nature, on 19 April. The study also describes the development of new molecules which when coupled with innovative instrumentation can enable simultaneous labeling and imaging of nearly 24 particular biomolecules. This number is almost five times the number of biomolecules that can be imaged simultaneously using prevalent techniques.

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. 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.

Wei Min, Associate Professor, School of Chemistry, Columbia University

Despite the fact that prevalent techniques for analyzing various structures in living cells and tissues have specific advantages, they are hampered by various fundamental drawbacks, a main drawback being the occurrence of a “color barrier.”

For instance, fluorescence microscopy is highly sensitive and is fundamentally the most common method adopted in biology labs. The microscope enables researchers to examine cellular processes in living systems by making use of “fluorescent proteins” which normally come in five colors. Each fluorescent protein has a target structure to which it applies a “tag,” or color. The five fluorescent proteins (or colors) generally used for tagging the structures are BFP (Blue Fluorescent Protein), mVenus (Yellow Fluorescent Protein), ECFP (Cyan Fluorescent Protein), DsRed (Red Fluorescent Protein), and GFP (Green Fluorescent Protein).

In spite of its advantages, fluorescence microscopy is hampered by the “color barrier,” which restricts the number of colors observed to a maximum of only five structures at an instant of time. This is due to the fact that the fluorescent proteins emit an array of indistinguishable shades that are consequently grouped into five broad color categories.

For instance, a researcher attempting to monitor the entire range of structures and disparate cell types in a live sample of brain tumor tissue will be restricted to monitoring just five or less structures at an instant of time on a single tissue sample. If the researcher wishes to monitor more than five structures, then the fluorescent labels used for identifying and tagging the last five structures have to be cleaned from the tissue to enable usage of the same fluorescent labels to recognize one more set of five or less structures. The researcher must repeat this procedure for every set of five or less structures to be observed. Apart from being labor intensive, the process can lead to loss or damage of crucial components of the tissue while cleaning the tissue.

“We want to see them all at the same time to see how they’re operating on their own and also how they're interacting with each other,” stated Lu Wei, postdoctoral researcher in the Min lab as well as the lead author of the study. “There are lots of components in a biological environment and we need to be able to see everything simultaneously to truly understand the processes.”

Apart from fluorescence microscopy, at present there are a range of Raman microscopy methods being used for monitoring living cell and tissue structures. These methods function by making the vibrations arising from characteristic chemical bonds in structures visible. Conventional Raman microscopy yields high-definition colors that fluorescence microscopy lacks, however at lower sensitivity. Fundamentally, better sensitivity mandates a concentrated, strong vibrational signal that can be attained only when there are millions of structures that have the same chemical bond. However, visualization of the associated structure might be almost impossible if the strength of the signal from the chemical bonds is poor.

In order to overcome this difficulty, Min and his colleagues — Chemistry Professor Virginia Cornish and Neuroscience Professor Rafael Yuste, used an innovative hybrid of prevailing microscopy methods.

They created an innovative platform known as electronic pre-resonance stimulated Raman scattering (epr-SRS) microscopy that integrates the advantages of Raman and fluorescence microscopy methods, thus leading to a high level of selectivity and sensitivity. The new method enables recognizing the structures with exceptional specificity as well as considerably lower concentration. Rather than using millions of the same structure for identifying the occurrence of that structure in conventional Raman microscopy, the new device necessitates just 30 structures for identification. The method also employs an innovative set of tagging molecules developed by the researchers to synergistically work with the ultramodern technique. The amplified “color palette” of molecules widens the tagging capacities, enabling nearly 24 structures to be images at the same time, instead of only five fluorescent colors. The researchers consider that the number of structures can be further increased in the future.

The researchers have victoriously investigated the epr-SRS platform in brain tissue. “We were able to see the different cells working together,” stated 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.” Wei further stated that the team is not limiting the usage of this technique to just the brain tissue. “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.”

According to Min, the innovative platform has various potential applications. He further added that the technique can be prospectively used for treating tumors that cannot be cured with prevalent drugs.

If we can see how structures are interacting in cancer cells, we can identify ways to target specific structures more precisely. This platform could be game-changing in the pursuit of understanding anything that has a lot of components.

Wei Min, Associate Professor, School of Chemistry, Columbia University