An interactive application for the measure of the intracellular Ca++ ion concentration in vital cells.
Copyright 1996-97 M.Pini and A. Rubini
Computer and Systems Engineering Department
University of Pavia
Medical Consultant Prof. F. Tanzi
General Physiology Department
University of Pavia
Recent advances in video camera and computer technology greatly enhanced the power of quantitative microscopy. In particular, it is now possible to measure the intracellular ion concentration in real time in vital single cells by using optical methods.
To do this job an epi-illumination fluorescent microscope, with a Xenon or mercury lamp, a wavelength changer, like a computer driven wheel holding some optical filters, and a light detector, usually a photometer or a video camera, are used. Finally a suitable optical probe (often called dye) is required, able both to bind the ion (more or less selectively depending on the dye) and, upon binding, to change its fluorescent characteristics: the greater the change in ion concentration, the greater the change in the emitted light.
The actual ion concentration may be computed by a calibration procedure, which is however, not very easy to do. The emitted light is not only function of the ion concentration but depends on other non specific factors, like the dye concentration and the thickness of the loaded cell. Further, the dye tends progressively to quench following light exposure. To prevent these difficulties, the so called radiometric method is used, where the ratio of the light intensity measured at two different excitations or emission wavelength is computed. The procedure reported by Grynkievicz et al. (1985) allows to obtain the Ca++ concentration.
The CFA system has been developed to measure the intracellular Ca++ concentration by using a graphical interface that allows to control the whole system parameters and to monitor the experiment evolution in real-time.
The Fura-2 dye is used, which is able, in the ester form, to go through the cell membrane but, after entering the cell and being hydrolyzed, becomes both sensitive to Ca++ and unable to cross the membrane back. The dye is excited at 380nm and 340nm, by using a Lambda-10 serial RS-232 controlled filter wheel, and the light emitted at 510nm is measured. If the Ca++ concentration increases, the light emitted when excited at 340nm increases, while the light emitted when excited at 380nm decreases. Even if UV light is used, there is no need for special suited optic. The 380nm excitation light was however reduced about 90% by a neutral density filter, to become comparable to the intensity of the 340nm light.
A high sensitivity video camera with a light-intensifier controlled via RS-232 serial-interface is connected to the microscope.
The system is controlled by a 90MHz Pentium computer with 16 Mbytes of RAM and 500 Mbytes HD running Linux 2.0.12 operating system that controls both the filter wheel and the camera intensifier.
Data are acquired through an ImageNation CX100 frame grabber and processed by using Pacco.
The CFA scheme.
The user interface of CFA allows complete control of all the system parameters via a graphical interface. When a new experiment is started a setup dialog appears allowing the researcher to set the camera and intensifier gain, the sampling time and the other acquisition parameters (filter wheel speed, false color analysis, ...). in real-time.
The CFA setup window.
After the setup is completed, three graphs showing the flow of the average intensity of each region of interest at 340nm, 380nm and the ratio are displayed.
The user can also show another window that monitor the distribution of Ca++ inside every cell by mean of a false color palette.
The CFA main windows.
The CFA program was developed for the Department of Physiology of the University of Pavia. Because of the mandatory hardware needed, the CFA program is not yet distributed via ftp. However if you want a copy of CFA or you want to ask me to support particular hardware. please mail to: firstname.lastname@example.org.