Microorganisms have been harnessed to produce a wide variety of products that can replace both petroleum-derived fuels as well as chemicals and polymer precursors derived from petroleum. Current approaches for studying cell metabolism are typically destructive, can perturb metabolic rates, such as through heating, or are only possible in vitro. Techniques for in situ and non-invasive monitoring of cell metabolism would transform understanding of the mechanisms and controls of designed microbial biosynthesis, as emphasized in the 2017 BERAC report Grand Challenges for Biological and Environmental Research: Progress and Future Vision. The goal of this proposal is to develop and use photon-avalanching upconverting nanoparticles – exceptional new optical probes developed at Columbia and LBNL – as microbial nanothermometers, offering the promise of seeing single-cell metabolic changes in real time, without significant phototoxicity or photodegradation, in response to either biosynthetic inputs or environmental conditions. Our recent breakthroughs of high-resolution near infrared (NIR) imaging of photon avalanching nanoparticles (ANPs) and reliable methods for targeting biocompatible nanoparticles to specific subcellular locales may now be leveraged for monitoring of metabolism in situ with deeply subcellular resolution. ANPs absorb multiple photons in the NIR and efficiently emit at higher energies in the NIR or visible regions, with no measurable photobleaching and no overlap with cellular autofluorescence. ANPs can be imaged deep into scattering environments and are up to a billion-fold more efficient than other anti-Stokes probes. ANPs exhibit the highest nonlinearities of any nanoscale material, which enable real-time NIR imaging at 70 nm resolution with just simple confocal microscopy. In addition, super-resolution analysis can produce sub-Angstrom localization accuracies. We are now showing that ANPs are sensitive to subtle changes in temperature and are pumped at NIR wavelengths that minimize sample or probe heating. The potential impacts of our objectives are far-reaching, directly addressing priority BER research areas including “super-resolution nanoscopy and sensing”, “imaging metabolic pathways”, and “advancing engineering of microorganisms”.The innovations will result in a radically simplified near infrared optical bioimaging system with far better resolution than is achievable with existing probes. Our research will establish foundational basic science that provides access to unprecedented detail for understanding and optimizing biosynthetic processes within engineered microbes. This work is critical for realizing the full potential of engineered biosynthesis and these novel nanomaterials, jump-starting the development of unparalleled society-impacting bioenergy and environmental technologies.
