Therefore, these three wavelengths must be fixed in the instrument's software. The excitation and emission slits must also be set. Depending on the machine and the lipid concentration, the instrument needs to be tuned to the optimal slit width to obtain the best signal to noise (S/N) ratio. Getting the highest possible fluorescence intensity is desirable to ensure high-resolution data is collected, but this intensity must not overshoot the maximum intensity the machine can record. Once these parameters (and others, depending on the machine) have been saved as 'the method' used to run an experiment, and the instrument was warmed up (the time required for a spectrofluorometer to warm-up is specific to the machine), the transport assays can be