3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30–50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

Cancer cells are usually found to be softer than normal cells, but their stiffness changes when they are in contact with different environments because of mechanosensitivity. For example, they adhere to a given substrate by tuning their cytoskeleton, thus affecting their rheological properties. This mechanism could become efficient when cancer cells invade the surrounding tissues, and they have to remodel their cytoskeleton in order to achieve particular deformations. Here we use an atomic force microscope in force modulation mode to study how local rheological properties of cancer cells are affected by a change of the environment. Cancer cells were plated on functionalized polyacrylamide substrates of different stiffnesses as well as on an endothelium substrate. A new correction of the Hertz model was developed because measurements require one to account for the precise properties of the thin, layered viscoelastic substrates. The main results show the influence of local cell rheology (the nucleus, perinuclear region, and edge locations) and the role of invasiveness. A general mechanosensitive trend is found by which the cell elastic modulus and transition frequency increase with substrate elasticity, but this tendency breaks down with a real endothelium substrate. These effects are investigated further during cell transmigration, when the actin cytoskeleton undergoes a rapid reorganization process necessary to push through the endothelial gap, in agreement with the local viscoelastic changes measured by atomic force microscopy. Taken together, these results introduce a paradigm for a new-to our knowledge-possible extravasation mechanism.

Cancer metastasis is a complex process involving cell-cell interactions mediated by cell adhesive molecules. In this study we determine the adhesion strength between an endothelial cell monolayer and tumor cells of different metastatic potentials using Atomic Force Microscopy. We show that the rupture forces of receptor-ligand bonds increase with retraction speed and range between 20 and 70 pN. It is shown that the most invasive cell lines (T24, J82) form the strongest bonds with endothelial cells. Using ICAM-1 coated substrates and a monoclonal antibody specific for ICAM-1, we demonstrate that ICAM-1 serves as a key receptor on endothelial cells and that its interactions with ligands expressed by tumor cells are correlated with the rupture forces obtained with the most invasive cancer cells (T24, J82). For the less invasive cancer cells (RT112), endothelial ICAM-1 does not seem to play any role in the adhesion process. Moreover, a detailed analysis of the distribution of rupture forces suggests that ICAM-1 interacts preferentially with one ligand on T24 cancer cells and with two ligands on J82 cancer cells. Possible counter receptors for these interactions are CD43 and MUC1, two known ligands for ICAM-1 which are expressed by these cancer cells.