Our current understanding of in vitro protein folding is due to decades of experimental and computational research that provided high-resolution characterization of protein structure, identification of folding principles, and development of folding algorithms. This research has benefited scientists broadly. For example, machine learning can now predict a protein’s 3D shape from its amino-acid sequence, proteins can be designed de novo for a specific fold or function, and supercomputers can simulate milliseconds of protein dynamics with all-atom resolution. With the success of in vitro studies, it is essential that we now turn our attention in vivo. While evidence that the cellular environment perturbs protein behaviors emerged over half a century ago, we still have limited fundamental information about the effects of these cooperative cellular interactions on protein properties. The gap in knowledge is largely attributable to the transient nature of interactions in the cellular milieu and challenges associated with studying protein structure, stability, and dynamics in living cells. Here we leverage groundbreaking spectro-microscopy methods, fast relaxation fluorescence imaging (FReI) and optical photothermal infrared imaging (O-PTIR) in combination with functional biochemical assays, in vitro biophysical spectroscopy, and numerical analysis solutions, to characterize in-cell protein dynamics. Our work addresses the following questions: 1. What is the physiochemical code to protein folding? Do classic in vitro protein principles translate to cells? 2. Can we develop methods to visualize the spatial distribution of metabolism and associated metabolic protein structural dynamics in living cells? 3. How does thermal adaptation and acclimation by organisms change the stability, folding, and aggregation of proteins in differentiated tissues? Overall, this work will lead to a greater understanding of protein homeostasis in health and disease.