Cell membrane capacitance is directly proportional to cell surface area. This is true because membrane composition, thickness and dielectric constant are invariant, and not changed by differences in membrane protein density. It is thus possible to monitor cellular capacitance fluctuations as a measure of cell surface area changes that occur with exo- and endo-cytosis events. Rokhan LLC, doing business as Neuroscience Tools, has recently developed and commercialized the Digital Clamp One (DCO), the first patch clamp amplifier in which the incoming signal is digitized and digitally processed to calculate and generate the command signal to the cell. Other commercially available patch clamp amplifiers are advertised as digital; have digital data outputs, and are controlled by a host computer so that they need have no knobs on the front panel. However, they still feedback and amplify the analog input to generate the analog command signal back to the cell. The DCO provides digitally processed feedback that allows switching seamlessly from conductance clamp, to voltage clamp to current clamp, among other advantages offered by its dynamic, digital feedback capabilities. In Phase I, we will add capability as a high resolution, high speed cellular capacitance monitor, displaying capacitance fluctuations in femtofarads to reveal small surface area fluctuations occurring during e.g. exo- or endo-cytosis in isopotential cells. The instrument will generate a sinusoidal stimulus signal input superimposed on the command DC signal to the cell, and employ an innovative algorithm in the time domain to calculate capacitance by fitting a sine wave with the same frequency as the stimulus to the acquired data. The expected advantage of this novel approach are a high signal to noise ratio and the ability to accurately monitor cell capacitance with fast time resolution even when membrane conductance is changing. Cellular capacitance may be measured as a point value in the picofarads range, using the conventional means of whole cell voltage clamping, but the measured value will be less than the actual whole cell capacitance unless the cell is isopotential, which is the case only in small and round cells. It has recently been shown that it is possible to obtain a much more accurate measurement of whole cell capacitance in non-isopotential cells using current clamping rather than voltage clamping (Golowasch, et. al., 2009). Further, it is proposed that the ratio of the two ways of measuring may give valuable additional information about the cell type under study. In Phase II, we will develop software to enable the DCO to provide a fast assessment of a cell's capacitance by both voltage and current clamping methods. The ratio between them is a heretofore mostly underappreciated metric for the basic cellular morphology of cells. It will aid in identification of cell type and morphological changes during development and plasticity and to characterize stem cell maturity.
The proposed project will introduce and validate a novel algorithm for faster, more accurate monitoring of cell capacitance fluctuations, which are directly proportional to cell surface area. Events of exocytosis or endocytosis cause fluctuations in cell surface area that occur with release or uptake of substances from or into cells. The use of a new time domain algorithm is made possible by the speed of our digital feedback clamping amplifier, the Digital Clamp One.
