Developing multiparametric fluorescence microscopy to connect metabolism with cellular manipulations - Project Summary: Correlating metabolic changes with external or internal stimuli and cellular manipulations in high spatial detail is important for understanding causation and correlation in biological systems. Especially, direct pixel-by-pixel correlation of multiple observable quantities, e.g., metabolism, diffusion of proteins, membrane fluidity, is difficult to observe simultaneously. Connecting these physical and biophysical behaviors across multiple imaging modalities can enable us to understand how inhibition of one property, e.g. changing the diffusion of the protein, can influence other properties such as metabolism or membrane fluidity. This type of correlation and causation-based simultaneous imaging in the same biological system is something that is lacking in current fluorescence imaging-based systems. Investigation of these properties in high resolution via confocal and multiphoton fluorescence microscopes enables us to interrogate cellular and subcellular details of these interactions. The goal of my research program is to create a framework and seamless workflow of imaging methodology where all of these fundamental properties and interactions can be quantified and correlated at the native resolution of the acquired image. To do so, we propose combining the phasor approach to Fluorescence Lifetime Imaging (phasor-FLIM), phasor approach to hyperspectral imaging (sp-phasor) and phasor approach to Fluorescence Correlation Spectroscopy (phasor-FCS). We will further create an imaging biophysics research program which combines single-molecule (SM) and ensemble fluorescence experiments in vitro and in solution to get a deeper detail at the fundamental questions arising from the molecular interactions. Most imaging approaches are focused on increasing resolution (either in time or space) for structural elucidation. Our approach towards imaging is fundamentally different because we are focused on understanding the functional aspects and quantifying physical properties that can be observed using imaging modalities. In the next 5 years we want to establish the principle of this multiparametric and multidimensional phasor imaging and apply them to various biological systems, both in cells and in tissues. Our future goal is to combine the analysis methods developed here with camera-based systems, including light sheet microscopy, and extend these approaches to a faster instrumentation and eventually to whole-organism imaging.
