Abstract
Ultra-precise stimulation solely to individual mitochondria, without any influence to the whole cell, is quite difficult by traditional biochemical reagents. In mitophagy research, the mitochondria and even the whole cell usually suffer irreversible and great damage caused by treatment with potent chemicals. In this protocol, we present the technical procedures of our developed noninvasive ultra- precise laser stimulation (UPLaS) technology, which introduces precise stimulation to individual mitochondria, to excite mitochondrial Ca 2+ (mitoCa 2+ ) oscillations, with little perturbation to mitochondrial membrane potential (MMP), or mitochondrial reactive oxygen species (mitoROS). The mitoCa 2+ oscillation by UPLaS was able to initiate the PINK1/Parkin pathway for mitophagy. This protocol has good potential to benefit researches on mitophagy and mitochondrial diseases. Graphic : Figure 1.Flowchart of the UPLaS technology.The femtosecond laser (1030 nm, 1 MHz, 220 fs) can stimulate individual mitochondria (1 μm 2 ) for a short period (0.1 s), whereas confocal microscopy (CM) provides continuous cell imaging to monitor molecular dynamics in real time, before and after UPLaS.
Keywords:
Ca 2+; Confocal microscope; Femtosecond laser; Mitophagy; PINK1; Parkin; Ultra-precise laser stimulation.