Combining STED and SML microscopies
V. Nägerl, J.B. Sibarita and K. Inavalli
We have developed and constructed a new instrument, which combines STED and single-molecule localization (SML) microscopy on a single microscope platform. STED is a coordinate-targeted super-resolution modality, which is ideally suited for imaging neuronal morphology, e.g. dendritic spines, using highly concentrated and diffusible dye molecules as marker of cellular volume. By contrast, SML (sptPALM, uPAINT), which is based on the detection of single emitters, provides information about the distribution and diffusion of single molecules, e.g. membrane-bound proteins like synaptic receptors or intra-spine proteins, e.g. PSD-95.
We have established the labeling strategy and spectroscopic conditions to repeatedly go back and forth between STED and SML acquisitions in live dissociated neurons maintained at physiological temperature. Moreover, we have extended the STED effect into the z-axis to achieve an isotropic gain in spatial resolution, using a combination of helical and annular phase plates, that create a doughnut and ‘bottle beam’, respectively, intensity distribution of the STED beam in the focal plane.