Mitochondria imaged with the ReScan Confocal (RCM) microscope
Nikon Ti2 with Okolabs incubator and PFS (Perfect Focus System)
CFI Plan Fluor 40x oil 1.3 objective
ReScan Confocal add-on (RCM) from Confocal.nl
Omicron 561nm 20mW laser
Hamamatsu Orca Flash 4.0 V3 camera
Framerate: 1 frame per 10 seconds
Laserpower: 1 microwatt at sample plane
Pixel dwell time: about 1 microsecond
Cell type: HO1N1 cells
Staining: Bacmam 2.0 Mitochrondria RFP (Invitrogen™ C10601)
Glass bottom 8-well IBIDI chamber
Prolong Live 1:100 (LIFE TECHNOLOGIES P36975)
Cost of microscope
ReScan Confocal add-on: <40k EUR
Cost of label: 10 EUR
As the movie clearly illustrates there is no sign of phototoxicity. Mitochondria are very mobile during the entire timelapse. Also mitochondrial fusion and fission can be observed. The captured cells undergo mitosis at the start of the movie, indicating they tolerate the imaging very well.
The observed decay in fluorescence intensity can be attributed to the fact that the labeling method is based on the Bacmam expression system. With each cell division the number of viral particles is approximately halved, therefore lowering expression.
This proves the strong point of the ReScan Confocal (RCM) add-on: sensitivity. Because of its unique camera based detection very low excitation energies can be used and still high signal to noise is preserved.