Correlative imaging using ions and single molecules
Prof. Aleksandra Radenovic
From the plethora of correlative imaging modalities, SR techniques were most frequently combined with electron microscopy to provide protein-ultrastructure relationships at nanometer-scale resolution. At the other forefront of methods development, scanning probe microscopy techniques aim to capture nanoscale topographical dynamic changes of cells under physiological conditions. To avoid membrane deformation and to provide a method that could unlock long-term monitoring of the biological processes, we recently implemented SICM. The method currently experiences vast leaps in performance due to instrument developments and the ability to fabricate capillaries below tens of nanometers reliably.
In contrast to AFM, SICM is truly non-contact, and represents the soft cell surface much more faithfully. In addition to providing accurate topographic imaging with nanometer resolution, SICM can be used to measure membrane stiffness surface charges and allows local delivery of material (e.g. fluorescent probes). The use of self-blinking dyes in SR microscopy permitted imaging conditions such as low laser excitation intensities and negligible bleaching that are ideal for live-cell imaging. In addition, the high SNR and photophysical properties of self-blinking dyes allow us to extend multiplane cross-correlation analysis to the 4th order using 8-plane volumetric imaging, achieving up to 29 planes. Finally, with a combined SICM-SR setup we demonstrate long-term correlated live-cell imaging.