A small chamber for making optical measurements on single living cells at elevated hydrostatic pressure

A 100-microliters chamber has been developed to facilitate the use of optical techniques for the study of living cells under conditions of high hydrostatic pressure and other environmental manipulations. The chamber attaches to the mechanical stage of an inverted microscope and is capable of sustaining cells under physiologic conditions over a range of hydrostatic pressure from 1 to 150 atmospheres (1 atm = 101.325 kPa). Cells are located directly on the pressure supporting window that forms the bottom of the chamber. This window is made from coverslip glass which is bonded to the chamber using an epoxy adhesive and is thin enough to permit ultraviolet epi-illumination using high magnification, high numerical aperture objectives. Electrodes for stimulating excitable cells are also located on this window. In operation, the chamber is installed in a custom stage mount which provides X, Y, Z, and rotational alignment of chamber contents with respect to the microscope's optical axis. This allows precise registration of the cell specimen with the image sensing devices attached to the microscope. Mechanical and optical alignment can be performed at any pressure over the working range of the chamber. The separate cover that seals the chamber is also fitted with a window to allow for simultaneous transillumination of the specimen. A high pressure liquid chromatography pump is used to continuously perfuse the chamber with a solution that is controlled for temperature, pressure, gas tension, and ionic composition.