AN AUTOMATIC MONITORING-SYSTEM FOR EPITHELIAL-CELL HEIGHT

This paper describes an automatic method to measure cell height (h) of epithelia grown as monolayers on transparent filter supports. Tissues are mounted in an Ussing-type chamber enabling solution exchange on b oth sides. The apical and basal side of the epithelial cells are marke d with fluorescent beads. The image of the fluospheres is captured wit h a video camera and processed by a computer-based video imaging syste m. One basal reference bead in a gelatin layer on the filter support a nd up to three beads attached at the apical surface are used to monito r changes in cell height of three cells simultaneously. The focusing o f the microbeads is done automatically by moving the objective with a piezoelectric device mounted on the nosepiece of the microscope. The a lgorithm fnr locating the bead is based on the changes in fluorescent light intensity emitted by the fluospheres. The method has an accuracy higher than 0.1 mu m and a time resolution as low as 6 s if measureme nts are restricted to one bead at the apical side. The method was test ed on artificial model systems and used to measure volume changes in r enal cultured epithelia (A6) after exposing the serosal surface to hyp otonic solutions and replacing cell-impermeable sucrose by an organic compound (glycerol) with a smaller reflection coefficient. Serosal hyp otonicity elicited a rapid volume increase followed by regulatory volu me decrease, whereas the organic compound replacement caused a steady increase in cell volume.