Development of a high-pressure microscope and its application to biological systems.

We have developed a novel microscope that enables to monitor biological objects under high hydrostatic pressure. The high-pressure optical cell can be used up to 200 MPa (~2000 bar) and it was mounted on a commercial inverted microscope to observe epi-fluorescent images of individual microtubules tethered to kinesin molecules on observation window of the high-pressure optical cell. Here, we report two experiments using this microscope. First, we observed the effect of pressure on the structure of microtubules. It was found that the microtubule depolymerized from the both ends even in the presence of 10muM Paclitaxel with increasing the pressure. The shortening velocity increased exponentially with the increment of the pressure. Second, we performed in Vitro motility assays in the presence of ATP molecules. The sliding velocity decreased with the increment of the pressure. We consider that these structural and functional perturbations are caused by penetration of water molecules into the intermolecular binding sites