| This protocol demands a wavelength scan, so we will use function 6 from the main menu, 'set scanning parameters'. Using the keypad set the scan to run from 300 to 420nm with a 2nm bandwidth at both slits and a 2nm step size. The time at each step should be set according to your sampling rate or the amplitude of the signals you expect (Longer integration times will obviously give you larger signals). Once the parameters are set, return to the main menu and select function 7 to run the scan. Check the controller is set to the program you have just set and also confirm that the number of cycles is continuous and no trigger signal is expected. Option 6 will run the scan, which will continue until indefinitely. To identify the emission peaks for Fura-2 in isolated cells we would first permeabilise the membrane then run the scan in high calcium conditions to obtain the spectra at maximum ion concentration. We then change the perfusing solution to low or nominally zero calcium conditions (e.g. using 10mM EGTA or BAPTA) and repeat the scan to obtain the spectra at minimum ion concentration. Comparison of the spectra obtained in each situation will allow identification of the two regions of maximum signal change and the isosbestic point. For more accurate determination of the emission peaks, the residual spectrum after quenching the dye should be recorded and subtracted from the traces obtained. |