Knutson – 2024

Ultrafast laser microscopy to study proteins and DNA. Fluorescent Lifetime Imaging: Approaches and Applications

We develop and exploit the techniques of ultrafast laser microscopy. We have developed state-of-the-art instruments that help us determine the local flexibility, size, and structure of protein or DNA complexes both in solution and inside living cells. For years, we have been developing 2-photon, lifetime(FLIM) and FCS microscopy methods to extend a detailed spectroscopic approach to the cellular level. We have, for example, recently developed an intracellular sensor for [O2] based on Myoglobin-mCherry chimeras, and we are using that construct with FLIM to map oxygen levels in various organelles, cells and tissues (and also oxygen gradient chambers for tumor simulation).  We couple that metabolic sensor with our prior work mapping the free/bound ratio of NADH in cells including cardiac mitochondria (2p lifetime).  We are now adding proteins with lifetime sensitive to met state and thus sense reactive species.  Using other microscopes sensitive to protein mobility, we have e.g. quantified the mobility of oncogene TFs in nuclei. We have collaborated with NHLBI/LCE in the creation of new fs CARS microscopes to study water permeation across vessel walls, and we continuously develop global analysis tools for imaging. We also collaborate on lifetime studies in cuvettes or the microscope to help characterize novel bright fluorescent aptamers.

Depending on their interests, summer interns will all be introduced to fluorescence instruments and image/data acquisition plus:

• Help test and exploit our novel fluorescence chimeras that sense intracellular [O2] levels.
• Help develop computer software to "globally" analyze either features and flexibility of proteins and DNA, FLIM-FRET of aptamers, or micro/nanoscopic image sets in cells.
• Exploit FLIM (Fluorescence Lifetime Microscopy) features to push STED/STAQ resolution below 500A, using new “minesweeper” analysis.