Recent advances in optical imaging technology, such as single-molecule spectroscopy, two-photon fluorescence microscopy and super-resolution imaging, have revolutionized our ability to study living systems at the microscopic level. New physical concepts and biological discoveries are emerging. The central goal of our research program is to discover novel principles of optical spectroscopy and microscopy, and apply them to address important questions in life sciences.

By merging the interface between physical chemistry and chemical biology, we have established two new bio-imaging platforms: bioorthogonal nonlinear Raman imaging and molecular-switch mediated fluorescence microscopy. From a spectroscopy point of view, vibrational-spectroscopy-based Raman microscopy and electronic-spectroscopy-based fluorescence microscopy are complementary approaches for interrogating molecular quantum states.

Nonlinear Raman Microscopy

Fluorescence microscopy is currently the most popular contrast mechanism employed in optical imaging. However, fluorescence imaging faces fundamental limitations for studying the vast number of small bio-molecules such as metabolites (e.g., amino acids), second messengers, neurotransmitters and drugs, because the relatively bulky fluorescent tags often destroy or significantly alter the biological activities of small biomolecules. Therefore, how to probe these vital species inside cells represents a grand challenge. We aim to develop a novel and general strategy that would enable an unprecedented ability to map out the distribution and to follow the dynamics of small bio-molecules in living systems.

Our work label these small molecules with bioorthogonal tags such as alkyne (CC) or stable isotopes including deuterium and 13C, and then image the labeled species in cells by stimulated Raman scattering (SRS) microscopy, a nonlinear vibrational microscopy originally developed by Freudiger, Min and Xie as a label-free technique. Such a strategic coupling of bioorthogonal tags with nonlinear vibrational microscopy, which we name bioorthogonal nonlinear Raman imaging, could do for small biomolecules what fluorescence imaging has done for larger species, extending the regime of light microscopy beyond the common fluorescence and label-free approaches. A broad range of exciting applications have been demonstrated possible (most are for the first time), such as imaging protein synthesis, protein degradation, DNA replication, RNA turnover, drug distribution, choline metabolism and glucose uptake in living cells, tissues and animals. New biological insights are emerging.

Label free SRS microscopy
SRS imaging with alkyne tags
SRS imaging with isotope labeling

Molecular-Switch-Mediated Novel fluorescence Microscopy

Fluorescence microscopy is the workhorse in modern biological imaging. While almost all existing fluorescence measurements exclusively rely on the bright state of the probes, one notable exception is the super-resolution technique that employs photoswitchable probes to break the diffraction barrier. Inspired by its success, we aim to exploit the relatively uncharted territory of photo-switching microscopy. An array of molecular-switch-mediated novel imaging principles have been discovered, including dark state imaging, genetically-encoded viscosity sensor, light-driven fluorescent timer, super-nonlinear fluorescence microscopy, and bioluminescence assisted switching and fluorescence imaging.

As diverse as these new principles appear, they all cherish a common notion and owe their core working mechanism to photo-switching spectroscopy (including singlet-triplet transition, cis-trans isomerization, photoconversion, photoactivation, stimulated emission, ground state depletion, etc). Together they have opened up a new dimension of light microscopy where molecular switching between quantum states not only breaks traditional physical limits (such as extending the fundamental imaging depth limit) but also enables new functional capabilities (such as timing the intracellular protein ages).