Table 2. Scanning parameters for performing FRAP analysis on mitochondria.

Parameter	Option A	Option B	Option C
Objective	63×	63×	63×
Zoom	1	2	2-3
Pixel size	512×80	512×512	512×512
Scan speed	9	8	7-8
Scan number for averaging	1	1	1
*Pinhole	Open	Open	Open
Laser intensity	0.5-10%	0.5-10%	0.5-10%
Detector gain	600	600	600
Size of ROI	0.5×0.5 μm	Variable#	Variable#
Time ineterval	5 msec	1s	5mins
Bleaching time	7μs	>1ms<1s	>1ms<1s
Bleach iterations	2	5	5
Time lapse imaging time	500ms	1-5min	30-1h

^*The pinhole should be kept open to be able to collect light from the whole depth of the cell. If the pinhole were narrow then movement of mitochondria in and out of the optical plane would introduce fluctuations in the reading. However, an open pinhole would compromise the resolution to a certain extent. Reduction of laser intensity can sometimes help to avoid out of focus signal, which, however, would depend on organization of mitochondria in the cell of interest.

^#Refer to the text on ROIs