Multiplexed Live-Cell Profiling with Raman Probes

Although single-cell multiparameter measurement has been increasingly recognized as a key technology toward systematic understandings of complex molecular and cellular functions in biological systems, it is still generally challenging for existing methods to decipher a large number of phenotypes in a single living cell, despite extensive efforts in analytical techniques. Wei Min, of the Department of Chemistry at Columbia University in New York City, and his associates recently published a paper outlining their devising a set of multiplexed Raman molecular probes with sharp and mutually resolvable Raman peaks to simultaneously quantify cell surface proteins, endocytosis activities, and metabolic dynamics of an individual live cell. Min, who recently spoke to us about this work, is the 2022 recipient of the Craver Award, presented annually at FACSS SciX to recognize the efforts of young professional spectroscopists that have made significant contributions in applied analytical vibrational spectroscopy. This interview is part of an ongoing series of interviews with the winners of awards that are presented at the annual SciX conference, which will be held this year from October 2 through October 7, in Covington, Kentucky.

What makes single-cell multiparameter measurement recognized as a key technology toward systematic understandings of complex molecular and cellular functions in biological systems?

Multiparameter measurement of single cells is important because of the immense structural and functional complexity of living systems. A recurring theme is that a large number of players (molecules) are interacting at every length scale, ranging from protein networks (10~100 nm), to organelles (100 nm – 1 µm), to various cell types within tissues (1~100 µm), to synergistic tissues within functional organs (100 µm – 1 cm), and reaching maximum complexity in nervous systems (~80 billion neurons, each interacting > 1000s of other neurons).

Your paper (1) describes the exploitation of combining ultra-bright Raman dots (Rdots) with simulated Raman scattering (SRS) microscopy as an alternative measurement technique for super-multiplex live-cell profiling. What allows Rdots combined with SRS to be an enhanced imaging technique over the use of fluorescent dyes?

Rdots are specially designed so that their spectral response are much sharper with higher resolution (50 times) than the standard florescent dyes. This allows us to employ many different “colors” of Rdots to target a large number of biological targets simultaneously.

Briefly discuss your overall findings and their implications. Were you surprised by the results?

After developing the technique, we used it for multiplexed live-cell profiling and phenotyping under various drug perturbations. We found that our single-cell multiparameter measurement can readily enable powerful clustering, correlation, and network analysis with biological insights. The results are surprisingly robust and hence we believe the technique can be applicable to a wide range of live cell assays that require multiplexed reading.

Are there any challenges or limitation involved with utilizing your technique? What options or alternative developments are available to overcome these challenges or to improve your approach?

Bioconjugation of Rdots to target specific biological species or pathways can be a potential challenge. The good news is that many protocols are available in the community, as people have accumulated enough experience working with other nanoparticle-based imaging reagents such as quantum dots and gold nanoparticles.

What sort of response has your paper received from the research community?

Many people in our field were surprised by the simplicity of the Raman microscope technique and the strong size of the signal brought by Rdots.

What are your next steps regarding this research?

In addition to multiplexed detection, which essentially uses one color for each target, we are also exploring a barcoded detection strategy which utilizes a combinatorial scheme to encode and then decode a large pool of similar objects (molecular mapping).

The Craver Award is presented annually to recognize the efforts of young professional spectroscopists that have made significant contributions in applied analytical vibrational spectroscopy. Please comment on the meaning of being 2022’s recipient.

I feel much honored to be recognized by the Craver award. In the field of Raman microscopy, label-free imaging has been the prevalent paradigm for decades. It is pleasing to see that our efforts of developing Raman-active imaging probes (including small tags, organic dyes and nanoparticles) are being accepted by the community. More and more researchers are now incorporating Raman probes into their research strategy, opening up more applications of vibrational spectroscopy in biomedical science and technology.

Reference

(1) C. Chen, Z. Zhao, N. Qian, et al., Nat Commun 12, 3405 (2021). https://doi.org/10.1038/s41467-021-23700-0

Wei Min is currently a Professor of Chemistry at Columbia University. He is also affiliated with the Department of Biomedical Engineering, the Kavli Institute for Brain Science and NeuroTechnology Center at Columbia University. After graduating from Peking University in 2003, he received his PhD from Harvard University in 2008 studying single-molecule biophysics with Prof. Sunney Xie. After continuing his postdoctoral work in Xie group, Min joined the faculty at Columbia University in 2010, and was promoted to Full Professor there in 2017.

Min has made important contributions to the development of stimulated Raman scattering (SRS) microscopy and employed it to open up a broad range of applications. He has co-invented modern SRS microscopy and pushed its sensitivity down to single molecule level through stimulated Raman excited fluorescence (SREF) spectroscopy; his team has designed and synthesized Raman-active probes exhibiting rainbow-like spectral “colors”; his group has opened up applications including bioorthogonal chemical imaging of small biomolecules (such as lipids, amino acids, glucose, and drugs), metabolic activity imaging in animals, and super-multiplexed vibrational imaging.