High-Speed Atomic Force Microscopy Imaging of DNA Three-Point-Star Motif Self Assembly Using Photothermal Off-Resonance Tapping.

High-speed atomic force microscopy (HS-AFM) is a popular molecular imaging technique for visualizing single-molecule biological processes in real-time due to its ability to image under physiological conditions in liquid environments. The photothermal off-resonance tapping (PORT) mode uses a drive laser to oscillate the cantilever in a controlled manner. This direct cantilever actuation is effective in the MHz range. Combined with operating the feedback loop on the time domain force curve rather than the resonant amplitude, PORT enables high-speed imaging at up to ten frames per second with direct control over tip-sample forces. PORT has been shown to enable imaging of delicate assembly dynamics and precise monitoring of patterns formed by biomolecules. Thus far, the technique has been used for a variety of dynamic in vitro studies, including the DNA 3-point-star motif assembly patterns shown in this work. Through a series of experiments, this protocol systematically identifies the optimal imaging parameter settings and ultimate limits of the HS-PORT AFM imaging system and how they affect biomolecular assembly processes. Additionally, it investigates potential undesired thermal effects induced by the drive laser on the sample and surrounding liquid, particularly when the scanning is limited to small areas. These findings provide valuable insights that will drive the advancement of PORT mode's application in studying complex biological systems.

Cancer-cell stiffening via cholesterol depletion enhances adoptive T-cell immunotherapy.

Malignant transformation and tumour progression are associated with cancer-cell softening. Yet how the biomechanics of cancer cells affects T-cell-mediated cytotoxicity and thus the outcomes of adoptive T-cell immunotherapies is unknown. Here we show that T-cell-mediated cancer-cell killing is hampered for cortically soft cancer cells, which have plasma membranes enriched in cholesterol, and that cancer-cell stiffening via cholesterol depletion augments T-cell cytotoxicity and enhances the efficacy of adoptive T-cell therapy against solid tumours in mice. We also show that the enhanced cytotoxicity against stiffened cancer cells is mediated by augmented T-cell forces arising from an increased accumulation of filamentous actin at the immunological synapse, and that cancer-cell stiffening has negligible influence on: T-cell-receptor signalling, production of cytolytic proteins such as granzyme B, secretion of interferon gamma and tumour necrosis factor alpha, and Fas-receptor-Fas-ligand interactions. Our findings reveal a mechanical immune checkpoint that could be targeted therapeutically to improve the effectiveness of cancer immunotherapies.

Mechanical Properties of Soft Biological Membranes for Organ-on-a-Chip Assessed by Bulge Test and AFM.

Advanced in vitro models called "organ-on-a-chip" can mimic the specific cellular environment found in various tissues. Many of these models include a thin, sometimes flexible, membrane aimed at mimicking the extracellular matrix (ECM) scaffold of in vivo barriers. These membranes are often made of polydimethylsiloxane (PDMS), a silicone rubber that poorly mimics the chemical and physical properties of the basal membrane. However, the ECM and its mechanical properties play a key role in the homeostasis of a tissue. Here, we report about biological membranes with a composition and mechanical properties similar to those found in vivo. Two types of collagen-elastin (CE) membranes were produced: vitrified and nonvitrified (called "hydrogel membrane"). Their mechanical properties were characterized using the bulge test method. The results were compared using atomic force microscopy (AFM), a standard technique used to evaluate the Young's modulus of soft materials at the nanoscale. Our results show that CE membranes with stiffnesses ranging from several hundred of kPa down to 1 kPa can be produced by tuning the CE ratio, the production mode (vitrified or not), and/or certain parameters such as temperature. The Young's modulus can easily be determined using the bulge test. This method is a robust and reproducible to determine membrane stiffness, even for soft membranes, which are more difficult to assess by AFM. Assessment of the impact of substrate stiffness on the spread of human fibroblasts on these surfaces showed that cell spread is lower on softer surfaces than on stiffer surfaces.