The microfluorometry is a sensitive method that makes it possible to measure [Ca²⁺]_i.from single living cells. The system consisted of an inverted epifluorescence microsope (Olympus CK 40).   The cell was studied using a 40×1.3 NA oil immersion objective (40× UV APO). Two lights of wavelengths 340 nm and 380 nm were produced by a monochromator from a white light source, and focused on the cells.

A coverslip was mounted on an open perfusion chamber that was designed for these types of experiments, and placed on the stage of the microscope. The physiological salt solution was superfused by closed loop flow system by means of peristaltic pump. A water bath and a thermistor connected to the perfusion chamber were used to control the temperature of the flowing physiological solution. The [Ca²⁺]^­­_i was measured by dual wavelength excitation fluorometry. The monochromator (PhotoMed DeltaRam) produces two excitation wavelengths 340 nm and 380 nm. The emitted light chosen by a 510 nm filter was detected by the photomultiplier tube detector and the signals fed to a computer containing the Felix32 software. The cells were excited at two wavelengths of light (340 nm and 380 nm) alternately. 340 nm signals correspond to Ca²⁺ bound Fura-2 molecules, and the 380 nm signals correspond to free Fura-2 molecules. In order to consider a [Ca²⁺]_i increase, the 340 nm signal should increase, and the 380 nm signal should decrease. For each second, one 340/380 ratio was obtained. The ratio between emitted fluorescence intensities of 340 nm and 380nm signals were calculated by Felix32 software. The corresponding [Ca²⁺]_i increase was elucidated from the calibration data.

The region of interest containing single cells was located with the help of diaphragm. The cell selected for experiment should be round, relatively big with sharp edge, and should posses a relatively high and stable fluorescent signal. The experiments were done in a dark room to avoid the interference of external light sources. The background fluorescence was measured by moving the focus away from the examined cell, and then subtracted from original fluorescence signal of 340 nm and 380 nm wavelengths, and a new ratio was calculated by using Felix32 software.