The perfect reference is a uniform fluorescing sample (e.g. solution of fluorophores), containing fluorophores that have a mono-exponential lifetime, are excited at the same wavelength and emit with the same wavelength as the sample, and do not bleach. We recommend 1 micromolar fluorescein solution pH 10 (4.00 ns) or erythrocine solution (86 ps) for GFP samples and 10 micromolar fluorescein solution pH10 (4.00 ns) for CFP samples.
Please make sure that your reference can be excited at the same excitation wavelength as your sample. Most ideal is to get similar intensity values for the reference stack as for the sample stack. It is highly recommended that you keep the MCP gain of the image intensifier and the LED DC current the same during the reference and sample acquisitions. To get similar intensity values, you can change the exposure time, use ND filters, or change the concentration of your reference solution.