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Center for Nonlinear Studies |
Recent advances in single-molecule fluorescence techniques yield access to quantitative and structural information in living and fixed cells in an unprecedented manner. Imaging of biomolecular structures with a resolution better than 20 nm with localization microscopy or other nanoscopy techniques has almost become routine in fluorescence microscopy. So far, most approaches make use of photo-physical processes to specifically switch fluorescent probes between their bright and dark states to improve resolution. Recently, we could show that reversible chemical reactions can also be used for stochastic activation and deactivation of fluorescent probes [1]. The probe system is based on a reversible complexation of copper(II) cations which allows controlling the proportion of molecules in the off/on-state by changing the copper(II) concentration. Currently, we investigate alternative reactions to learn to which extend such probes can be used in microscopy. Along that way we also study associated chemical reactions at single-molecule level. Besides probe development, we are also interested in exploiting the phenomenon of photon-antibunching for quantitative analysis of fluorescently labeled samples [2, 3]. Here, we could experimentally demonstrate that calibration-free counting of emitting molecules can be achieved reliably for up to 18 molecules with an error of about 30%. We can now apply this method to directly asses and compare the number of labels of arbitrary probes, like DNA-oligonucleotide and/or proteins to characterize labeling stiochiometry and efficiency which is an essential prerequisite for quantitative studies in biological samples
Host: Doug Shepherd, MPA-CINT and CNLS, dpshepherd@lanl.gov