1.1	Turn ON confocal system.
1.2	Place a specimen on the stage with the coverslip side facing the objective.
1.3	Focus on the outer surface of the coverslip while looking through the eyepieces. This may be achieved either in fluorescence mode, using the Hg or Xe lamp provided or using transmission light. If the specimen contains rare and sensitive staining, focus on an area on the coverslip away from it.
1.4	Select the objective and the lasers that will be needed for the imaging experiment. You will typically need as many lasers as fluorescent dyes. Most commonly used laser excitation lines are 405 nm for blue emitters, 488 nm for green, 543 nm or 561 nm for red and 633 nm for far red.
1.5	Select reflection mode. In the Leica AOBS, this is an option on the AOBS control interface. For other systems, it is sufficient to select dichroic and emission filters that allow the incident light to go through and reach the detector. The test may be run in multichannel mode, simultaneous testing as many laser lines as there are detectors.
1.6	Start scanning in live (Leica) or continuous mode. Start with detector gain values around the middle of the range, approximately 600 V.
1.7	Focus until the reflected image is the brightest. In the process the image may become saturated. In this case, reduce the detector gain until intensities fall around the middle of the intensity range, at around 130 for 8 bit detection (0–255).
1.8	Repeat for each laser of interest. This is only necessary when setting up the experiment. In all subsequent imaging days, the parameters saved in Step 1 will be used without adjustment.
1.9	Change mode to xzt, or vertical time lapse. In live mode, this should result in at least one bright horizontal line. Depending on the type of specimen, there may be two lines, the first and brightest is the reflection off the coverslip and the second, off the slide. For thick specimen the second reflection may not be in the field of view. For coverslip bottom chambers there will be, of course only one line.
1.10	Select a time interval of 30 s and acquire minimum 10 images, for a total of 5 min.
1.11	Save all instrument setting to be reused at least once a day, before starting the actual imaging experiments.
Caution	Make sure the intensity of the reflection line is not saturated.
Tip	Several laser lines may be tested simultaneously, as many as the number of detectors available on the system.