Correlative Stimulated Emission Depletion and Scanning Ion Conductance Microscopy.

Abstract

Correlation microscopy combining fluorescence and scanning probe or electron microscopy is limited to fixed samples due to the sample preparation and nonphysiological imaging conditions required by most probe or electron microscopy techniques. Among the few scanning probe techniques that allow imaging of living cells under physiological conditions, scanning ion conductance microscopy (SICM) has been shown to be the technique that minimizes the impact on the investigated sample. However, combinations of SICM and fluorescence microscopy suffered from the mismatch in resolution due to the limited resolution of conventional light microscopy. In the last years, the diffraction limit of light microscopy has been circumvented by various techniques, one of which is stimulated emission depletion (STED) microscopy. Here, we aimed at demonstrating the combination of STED and SICM. We show that both methods allow recording a living cellular specimen and provide a SICM and STED image of the same sample, which allowed us to correlate the membrane surface topography and the distribution of the cytoskeletal protein actin. Our proof-of-concept study exemplifies the benefit of correlating SICM with a subdiffraction fluorescence method and might form the basis for the development of a combined instrument that would allow the simultaneous recording of subdiffraction fluorescence and topography information.