A proof-of-concept study of combined scanning ion conductance and stimulated emission depletion microscopy
Philipp Hagemann, Astrid Gesper, Patrick Happel
Ruhr University Bochum, RUBION, Universitätsstr. 150, 44801 Bochum
Understanding plasma-membrane related processes such as endo- or exocytosis or cell migration requires simultaneous observations of the plasma membrane dynamics and changes in the distribution of proteins of interest. Since the plasma membrane exhibits a three-dimensional ultrastructure with features with a size in the range of nanometers, a resolution beyond the diffraction limit of light is desirable.
To record physiological membrane dynamics reliably, the impact of the imaging tools on the sample have to be minimized. A bias-free method for measuring the membrane topography of living cells is scanning ion conductance microscopy (SICM). To sense non-conducting surfaces like the cell membrane, it utilizes the ion current through the opening of an electrolyte-filled glass capillary. Because SICM can be operated under physiological conditions, it is suitable for long-term studies of membrane dynamics.
Here we present the first proof-of-principle recordings of correlated SICM and stimulated emission depletion (STED) microscopy. We recorded a STED image of actin and determined the nanostructure of the same, fixed HeLa cells with SICM. This is a first step towards the development of a combined STED-SICM instrument, which would provide the advantages of both techniques and allow investigating changes in topography and protein distribution in living cells with sub-diffraction resolution.