New Dimensions and Domains for Fluorescence Fluctuation Imaging Methods

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Advancing quantitative image correlation methods to the third dimension and beyond***In the past decade there has been a revolution in light microscopy recognized with the awarding of the 2014 Chemistry Nobel Prize to Betzig, Moerner and Hell for the development of super resolved fluorescence microscopy. This technical renaissance has witnessed tremendous advances in both super-resolution fluorescence microscopy and in single plane illumination microscopy (SPIM) also commonly known as light sheet microscopy. As was the case at the advent of light microscopy in the 17th century, these new technological leaps offer great promise for biomedical research science on living cells and organisms. However, many forms of super-resolution microscopy are based on iterative molecular localization leading to a kinetic bottleneck where temporal resolution is sacrificed to gain spatial resolution. ***This proposal is based on bridging this gap by developing new quantitative fluorescence fluctuation analysis tools suitable for super-resolution and light sheet microscopy methods. If a picture is worth a thousand words, a quantitative understanding of the dynamics of the molecular machines of life inside the cell is even more valuable especially as Biology needs to move toward a new quantitative basis in the post Genomic era. NSERC-DG funding has been fundamental in allowing us to develop new biophysics image analysis tools that unlock information on the transport properties and interactions of molecules imaged with standard fluorescence microscopy methods. These image correlation methods rely on a type of noise analysis of fluorescence fluctuations in the images which report on how the molecules imaged in the cell are behaving. By correlating the signals in space & time, we can reveal how quickly the fluorescently tagged molecules are moving, whether they are diffusing randomly or moving in a directed fashion, measure numbers of molecules in a given state, and report on the binding & interactions of molecules. We have also extended our method toolkit to include wavelet analysis at multiple scales which is especially suited for tackling the more difficult cell shapes encountered for cells growing in more realistic model 3D systems and in the tissues of living organisms. This proposal is centered on extending our image correlation, fluorescence fluctuation and wavelet tools to measure biomolecule dynamics from super-resolution microscopy measurements and in 3D applications for receptor cycling in cells and finally to measure collagenase enzyme kinetics maps in 3D for model cancer cells migrating in more realistic 3D collagen matrices. This DG program is built on a firm foundation of demonstrated excellence in quantitative fluorescence method development, microscopy/optics and completely new lab instrumentation in 3D light/lattice sheet microscopy and all major forms of super-resolution microscopy.