Michelle Digman, Department of Biomedical Engineering, University of California, Irvine
Title: “Advances in Image Correlation Microscopy and Metabolic Imaging”
DATE: Friday, February 1, 2019
LOCATION: Genomics Auditorium, RM 1102A
Host: Dr. Venugopala Gonehal
Fluorescence fluctuation spectroscopy has evolved from single point detection of molecular diffusion to a family of microscopy imaging correlation methods (i.e. ICS, RICS, STICS, and kICS) useful in deriving spatial-temporal dynamics of proteins in living cells. The advantage of these imaging techniques is the simultaneous measurement of all points in an image fast and sensitive cameras coupled to new microscopy modalities such as the sheet illumination technique. An emerging method in this area is being able to obtain diffusion routes adopted by proteins in the 2 and 3 dimensions. In this talk, I will discuss the evolution of fluctuation analysis from the single point source to mapping diffusion in whole cells and the technology behind this technique. The pair correlation fluctuation (pCF) analysis is done between adjacent pixels. We have expanded this method to calculate the correlation between pixels at a given distance in twenty four radial directions to provide a detection window into anisotropic molecular diffusion. Similar to the connectivity atlas of neuronal connections from the MRI diffusion tensor imaging this new tool will be used to map the connectome of protein diffusion in living cells. For biological reaction-diffusion systems, live single cell spatial-temporal analysis of protein dynamics provides a mean to observe stochastic biochemical signaling in the context of the intracellular environment which may lead to better understanding of cancer cell invasion, stem cell differentiation and other fundamental biological processes.
There are few methods to non-invasively monitor metabolic changes of individual living cells. The advantage to single cell characterization is that it can provide valuable information of underrepresented cell subpopulations that could have a differential function within the tumor mass. We have developed the phasor analysis, for hyperspectral resolved imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) as a tool used to identify multiple species. For spectral analysis each pixel in the image is used to construct a spectral profile that is Fourier transformed to produce the coordinates of the pixel in a polar plot. The relative fraction of endogenous free and bound NADH is a useful method for monitoring changes in energy metabolism since the NADH/NAD+ pair is crucial for electron transfer through the mitochondrial electron transport chain. The phasor spectral and FLIM analysis can provide a label-free, fit-free and sensitive method to identify different metabolic states of cells.