Pfltigers Arch (1993) 425:164-171 E6[ hfin Journal of Physiology 9Springer-Verlag1993 Instruments and techniques An automatic monitoring system for epithelial cell height Willy Van Driessche, Patrick De Smet, Gert Raskin Laboratory for Physiology, KU Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium Received March 23, 1993/Received after revision and accepted May 7, 1993 Abstract. 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 both sides. The apical and basal side of the epithelial cells are marked with fluorescent beads. The image of the fluospheres is captured with a video camera and pro- cessed by a computer-based video imaging system. One basal reference bead in a gelatin layer on the filter sup- port and up to three beads attached at the apical surface are used to monitor changes in cell height of three cells simultaneously. The focusing of the microbeads is done automatically by moving the objective with a piezo- electric device mounted on the nosepiece of the micro- scope. The algorithm for 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 gm and a time resolution as low as 6 s if measure- ments are restricted to one bead at the apical side. The method was tested on artificial model systems and used to measure volume changes in renal cultured epithelia (A6) after exposing the serosal surface to hypotonic solutions and replacing cell-impermeable sucrose by an organic compound (glycerol) with a smaller reflection coefficient. Serosal hypotonicity elicited a rapid volume increase followed by regulatory volume decrease, whereas the organic compound replacement caused a steady increase in cell volume. Key words: Cell volume - A6 epithelia - Osmolal- ity - Regulatory volume decrease - Fluorescent mi- crobeads - Ussing-type chamber - Microscopy Introduction During the last decade the response of cellular volume to changes in the composition of incubation media has Correspondence to: W. Van Driessche been the subject of numerous studies. The regulatory processes involved in adjusting volume after swelling cells with hypotonic media have especially challenged many investigators. The involvement of electroneutral as well as electrical conductive transporters allowing K + and C1- exit after cell volume expansion has been dem- onstrated. The activation and involvement of several ion translocators has been demonstrated by many labora- tories with electrophysiological techniques [18] and tracer experiments [7]. The decrease of cellular ion con- centration induced by hypotonic stress is followed by an osmotically driven water flow resulting in cellular vol- ume decrease. The time course of volume changes after challenging cells with hypo- or hypertonic media has been investigated with different methods: high-resol- ution three-dimensional microscopic video imaging [16], Coulter counter technology [11], measurements of cellu- lar 3H20 in combination with [14C]polyethylene glycol as extracellular marker [15], and concentration changes of fluorescent probes trapped in the cellular compart- ment [3]. Most methods require the use of non-attached cells such as nucleated red blood cells, lymphocytes, and Ehrlich ascites tumor cells. On the other hand, the regu- lation of cellular volume of intact epithelia is of con- siderable physiological interest. Indeed, transepithelial movements of salts and water can cause substantial alter- ations of cell volume as a consequence of transient im- balances of fluid flow into and out of the cellular com- partment [4]. Moreover, epithelial ceils are exposed to varying osmolalities as, for example, in the renal medul- lary segment of the kidney [1]. Regulatory mechanisms will mostly restore epithelial cells to their original vol- ume. Recently Crowe and Wills [2] described a simple method for recording changes in epithelial cell height. This technique utilizes fluorescent microbeads as high- contrast landmarks for the apical and basal cell surfaces. Cell height was measured by focusing on the apically and basally located microbeads and determining their spatial distance. The major advantage of this method is that volume changes are measured in intact epithelia