I will discuss two recent projects to extend and apply single-molecule spectroscopy and imaging in new regimes. The first demonstrates progress toward super-resolution fluorescence microscopy at cryogenic temperatures with single molecules. This technique offers the possibility of enhanced localization precision due to reduced photobleaching rates at low temperatures and the acquired images can be correlated with cryogenic electron microscopy data. Super-resolution microscopy requires dense fluorescent labelling and active control over the emissive state of the labels to achieve the required sparsity of emitters. Maintaining active control through blinking or photoactivation at cryogenic temperatures has proved challenging. However, we have identified a red photoactivatable protein, PAmKate, which remains activatable at 77 K with an efficiency comparable that at to room temperature. In initial cryogenic super-resolution experiments labelling PopZ, a protein known to localize to the poles of the model bacterium Caulobacter crescentus, we find considerable enhancement in the number of collected photons per PAmKate protein. This increased photon collection leads to a corresponding improvement in localization precision by more than a factor of four, with some proteins achieving sub-nanometer precision. (This work was done in collaboration with Annina Sartor, Jiarui Wang, Dr. Saumya Saurabh, Professor Lucy Shapiro, and Professor W.E. Moerner) The second project utilizes the Anti-Brownian Electrokinetic (ABEL) trapping device to simultaneously measure fluorescence lifetime, intensity, polarization, and spectrum from single complexes in solution. It was previously known that the orange carotenoid protein (OCP) interacts with the light-harvesting antenna structures in cyanobacteria (phycobilisomes) under high-light exposure to quench excess excitations absorbed by the phycobilisome. Our single-molecule multiparameter data reveals that the quenched phycobilisomes can be separated into two distinct populations, one less quenched and one more quenched. Comparison of these populations to simulations suggests they represent phycobilisomes with one or two bound OCPs, respectively. (This work was done in collaboration with Dr. Allison Squires, Dr. Nikki Magdaong, Dr. Haijun Liu, Professor Robert Blankenship and Professor W.E. Moerner)

Developments in cryogenic single-molecule super-resolution imaging and dynamics of photosynthetic antennas in solution

2019 Spring National Meeting

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