Volume Regulation and Non-Osmotic Volume of Individual Human Platelets Quantified by High-Speed Scanning Ion Conductance Microscopy.

Platelets are anucleate cells that play an important role in wound closure following vessel injury. Maintaining a constant platelet volume is critical for platelet function. For example, water-induced swelling can promote procoagulant activity and initiate thrombosis. However, techniques for measuring changes in platelet volume such as light transmittance or impedance techniques have inherent limitations as they only allow qualitative measurements or do not work on the single-cell level. Here, we introduce high-speed scanning ion conductance microscopy (HS-SICM) as a new platform for studying volume regulation mechanisms of individual platelets. We optimized HS-SICM to quantitatively image the morphology of adherent platelets as a function of time at scanning speeds up to 7 s/frame and with 0.1 fL precision. We demonstrate that HS-SICM can quantitatively measure the rapid swelling of individual platelets after a hypotonic shock and the following regulatory volume decrease (RVD). We found that the RVD of thrombin-, ADP-, and collagen-activated platelets was significantly reduced compared to non-activated platelets. Applying the Boyle van't Hoff relationship allowed us to extract the non-osmotic volume and volume fraction on a single-platelet level. Activation by thrombin or ADP, but not by collagen, resulted in a decrease of the non-osmotic volume, likely due to a release reaction, leaving the total volume unaffected.