• Are you using the right fluorescence filter cube?
  • Are you using the right reference solution and reference lifetime for calibrating the system? Did you change anything in the optical setup between taking a Reference and a Sample? Please note that each optical setup requires its own reference calibration; i.e. switching dichroics, filters, objectives, light source, magnifications, or power light levels changes the path lengths inside the system. The same holds when using a different MCP gain for the intensified CCD camera. Changing ND filters or the exposure time does not require a different reference calibration.
  • Is there reason to believe that the (photophysics of) your sample is as expected?
  • Is there auto-fluorescence influencing the fluorescence lifetime distribution?
  • In case your application is FRET; is there leak-through from your acceptor in the donor channel?