A SMALL CHAMBER FOR MAKING OPTICAL MEASUREMENTS ON SINGLE LIVING CELLS AT ELEVATED HYDROSTATIC-PRESSURE

A 100-mu l chamber has been developed to facilitate the use of optical techniques for the study of living cells under conditions of high hyd rostatic pressure and other environmental manipulations. The chamber a ttaches to the mechanical stage of an inverted microscope and is capab le of sustaining cells under physiologic conditions over a range of hy drostatic pressure from I to 150 atmospheres (1 atm = 101.325 kPa). Ce lls are located directly on the pressure supporting window that forms the bottom of the chamber. This window is made from coverslip glass wh ich is bonded to the chamber using an epoxy adhesive and is thin enoug h to permit ultraviolet epi-illumination using high magnification, hig h numerical aperture objectives. Electrodes for stimulating excitable cells are also located on this window. In operation, the chamber is in stalled in a custom stage mount which provides X, Y, Z, and rotational alignment of chamber contents with respect to the microscope's optica l axis. This allows precise registration of the cell specimen with the image sensing devices attached to the microscope. Mechanical and opti cal alignment can be performed at any pressure over the working range of the chamber. The separate cover that seals the chamber is also fitt ed with a window to allow for simultaneous transillumination of the sp ecimen. A high pressure liquid chromatography pump is used to continuo usly perfuse the chamber with a solution that is controlled for temper ature, pressure, gas tension, and ionic composition.