Super-multiplex optical imaging: development of novel spectroscopy and probes to illuminate complex biomedicine

Biological systems are inherently heterogeneous in space, dynamic in time, and complex in nature. A grand challenge is how to study the vast number of interacting players at every relevant length scale, ranging from protein network in protein complexes, to interacting organelles within cells, to various cell types within tissues, and to synergistic tissues within functional organs. Hence, the ability to simultaneously monitor a large number of interacting species inside biological systems with sufficient spatial-temporal resolution is indispensable for characterization and understanding of the underlying complexity. However, there is currently no suitable technology that can meet this grand challenge. The prevalent “omics” technologies do not have the required spatial-temporal resolution, especially for three-dimensional samples or living specimen. Optical microscopy can only image a few (2~5) different targets at once, limited by the fundamental “color barrier” of fluorescence. To break the color barrier of light microscopy and to bridge the gap between “omics” and imaging, here we propose a radically new technology platform. Novel vibrational spectroscopy including electronic-pre-resonance stimulated Raman scattering (epr-SRS) and stimulated Raman excited fluorescence (SREF) will be exploited, to achieve the most sensitive Raman imaging to date. Our preliminary data have proved single-molecule sensitivity. We will further develop the technique by exploring the two-dimensional excitation spectroscopy to reach ~100 colors, designing and synthesizing a library of imaging probes, opening up super-resolution super- multiplex imaging. The imaging technology will then be implemented in several broad-impact applications including super-multiplex tissue pathology, mapping brain-wide architecture complexity, and super-multiparameter deep phenotyping of living cells. Innovations in optical microscopy have changed the way many biological problems are addressed. Just like confocal microscopy is the work-horse in biomedical labs and two-photon fluorescence microscopy has transformed in vivo brain imaging, we envision our newly proposed super-multiplex spectroscopy and microscopy will break the current technical bottleneck, revolutionize multicolor optical imaging, and become a new standard for system-wide study of complex systems in general.