Functional Bond-Selective Microscopy: Physically Informed Insights in Chemistry and Biology

Biological systems are immensely complex, comprising diverse molecular species organized across multiple spatial and temporal scales. Through intricate networks of tightly regulated chemical reactions, cells are able to maintain homeostasis, respond to environmental stimuli, and meet energetic demands. Despite massive technological advances spanning many decades, a significant gap still exists in our understanding of how biochemical networks are locally regulated in living systems. Fundamental physicochemical parameters such as concentration, pH, temperature, and electrostatics, which ultimately govern chemical reactivity, remain challenging to image quantitatively, particularly in live cells. We argue that developing methods capable of quantitatively imaging these physicochemical parameters is not only essential for interrogating the regulation of biochemical reaction networks but also provides a rich framework for uncovering hidden, physically informed insights in chemistry and biology. To image chemical reactions in a biological context is a highly demanding task; few methods exhibit the necessary biocompatibility, spatial resolution, sensitivity, molecular specificity, and quantitative reporting capacity to access subcellular chemical information. Among existing optical methods, vibrational spectroscopy provides a natural context for understanding chemical reactions in cells, since molecular vibrations serve as chemical "fingerprints", reporting quantitatively on the formation, breaking, and interactions of chemical bonds. Despite this enormous potential, conventional vibrational imaging faces significant challenges in quantifying chemical kinetics and dynamics in cells. Endogenous biomolecules exhibit highly overlapped, convoluted spectra, largely prohibiting specific molecular identification. Moreover, conventional techniques, generally based on infrared (IR) absorption or Raman scattering, have limited sensitivity, and in the case of IR absorption, coarse spatial resolution while operating at the diffraction limit, obscuring subcellular detail. As such, there is a significant need for vibrational spectro-microscopies with improved molecular specificity, sensitivity, and spatial resolution. Coupling novel nonlinear optical strategies, which provide higher sensitivity and spatial resolution, with engineered vibrational probes, which afford higher molecular specificity and functionality, has proven fruitful in addressing these longstanding challenges in vibrational imaging and enabling functional bond-selective imaging. In this Account, we highlight recent work from our group toward quantitative measurements of physicochemical observables in biological systems. We first demonstrate the utility of small vibrational probes (deuterium labeling and alkyne tagging) with stimulated Raman scattering (SRS) microscopy for absolute concentration determination in protein aggregates and local pH sensing via hydrogen-deuterium exchange. We then discuss the development of two new imaging modalities, termed Boltzmann-edge vibrational thermometry (BET) and bond-selective fluorescence-detected IR-excited (BonFIRE) spectro-microscopy, and we highlight the applications of these methods toward quantitative measurements of local temperature and local electric fields. In each case, the pursuit of quantitative, physics-informed environmental reporting served as a launching point for the discovery of new biological or chemical phenomena. We believe that this is a valuable, unifying framework for understanding these works and provides a useful perspective toward the design of future experiments.