Achieving 15 nm resolution using localization with stimulated emission depletion microscopy (LocSTED)

Abstract

Both the branches of high resolution nanoscopy coordinate targeted and coordinate stochastic methods discern closely spaced molecules based on the same principle switching of fluorescent molecules between bright and dark states. When stimulated emission depletion (STED) microscopy uses a coordinate targeted approach and readout by applying a spatially modulated depletion pattern to switch off the molecules, single molecule localization microscopy (SMLM) uses a stochastic approach and readout by temporally switching a subset of fluorophores at a time. This thesis introduces localization with STED microscopy (LocSTED), a correlative approach combining STED microscopy and localization microscopy. This optical scheme allows determining the position of a single emitter in space using the simplest form of a STED microscope that uses a combination of continuous waves for both excitation and depletion. Stochastic reversible blinking of fluorophores is utilized within a predefined coordinate which is nanoscopic in size. The imaging was performed in a subdiffractional extent thus limiting the scanning field to the donut minimum. The potential of the method was shown by imaging fluorescently labeled proteins attached on nanostructures fabricated by STED lithography and also by imaging DNA origami nanorulers. Photobleaching as an irreversible reaction is usually a hurdle in STED microscopy because of the higher intensity of depletion beam used. An imaging buffer containing an enzymatic oxygen scavenger system and reducing and oxidizing system (ROXS) was developed and applied. ROXS renders STED more efficient. With a simple CW STED microscope, this thesis shows that single molecules can be localized with a localization error of 5 nm. By imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography, it is shown that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm. This shows that the classical resolution of a confocal microscope is improved to /35 and that of a CW STED microscope by a factor of at least 4. LocSTED microscopy thus allows estimating the total number of proteins attached on a single nanoanchor by enhancing the resolution of a simple CW STED microscope.submitted by Sujitha PuthukodanJohannes Kepler University Linz, Dissertation, 2019OeBB(VLID)454336