Bio-orthogonal Mid-Infrared Photothermal Imaging of Cancer Metabolism - Project Summary Understanding how cancer cells reprogram their metabolism and interact with other biological processes to gain aggressiveness and/or resistance to a treatment is of paramount significance to advancing current diagnosis and treatment strategies. Cancer metabolism is a highly complex and heterogeneous process, subject to micro- environmental cues. Investigating the vastly complex metabolic landscape of cancer remains extremely challenging due to intra-tumoral heterogeneity, cancer cell interactions with different cell types, and dynamic state of the microenvironment. Metabolomics has been a golden standard for detection of cellular metabolites. The disadvantages of metabolomics remain its destructive nature, prohibiting live cell or intravital analysis, and lacking spatial information. Fluorescence microscopy offers high selectivity and provides subcellular resolution. However, the fluorophore labels are much larger than the targeted metabolites (e.g. fatty acids, choline) and thus can hinder or alter the intrinsic metabolism. Here, we propose to fill in this gap through a new approach termed bio-orthogonal mid-infrared photothermal (MIP) imaging. By using a visible beam to sense the IR absorption induced refractive index change, MIP microscopy allows highly sensitivity imaging of chemical bonds at the visible diffraction limits. We propose to develop nitrile-based IR-active probes that produce extremely strong MIP signals in the spectrally silent region and induce minimal perturbation to the molecule of interest. In feasibility studies, we have developed a series of nitrile-based substrates that sense the activities of specific enzymes including caspase and phosphatase. Importantly, due to the much larger infrared absorption cross section than Raman scattering, the MIP limit of detection of these nitrile sensors reaches a few micromolar level, which is ~1000 times below the detection limit of alkyne-based Raman tags. On the instrumentation side, we have developed a laser-scan MIP microscope, which increased the imaging speed from millisecond per pixel to microsecond per pixel. Based on these preliminary studies, we propose to systematically develop bio-orthogonal mid-infrared photothermal imaging in three steps. (1) Building a library of nitrile chameleons to sense important molecular events inside cancer cells. (2) Building a library of nitrile-tagged small molecules to sense nutrient metabolism in cancer cells. (3) Developing microsecond-scale mid-infrared heating spectroscopy to enable multiplex bio-orthogonal imaging. Ovarian cancer cells will be used as a testbed. Our technology synergistically couples two innovations in probe development and instrumentation. Broad use of this technology, via dissemination and commercialization, will allow researchers to quantitatively measure various molecular events at subcellular level, and will open exciting opportunities for molecule-based precision diagnosis and discovery of novel targets for precision treatment of cancers and other diseases.
