Background

Precise stimulation of individual mitochondria, without perturbation to the whole cell or damage to mitochondria, is of great significance to mitochondrial research (Russell et al., 2020). Moreover, the isolated modulation of mitochondrial membrane potential (MMP), mitochondrial reactive oxygen species (mitoROS), or mitochondrial Ca²⁺ (mitoCa²⁺), without perturbation to the other two, might provide more insights into mitochondria function. Mitophagy is abnormal in Parkinson's disease (PD) (Liu, J. et al., 2019; Ge et al., 2020). The mechanism of mitophagy and its dysregulation is essential to the better understanding of PD. In previous works, where PINK1/Parkin mediated mitophagy has been studied (Jin and Youle, 2013; Lazarou et al., 2015; Pickrell and Youle, 2015; Chung et al., 2020), carbonyl cyanide m-chlorophenyl hydrazine (CCCP) was extensively used to depolarize mitochondria and trigger mitophagy. However, the completely depolarization of MMP and significant high-level mitoROS are irreversible, and prevent further investigation of the mechanism of action of mitochondria.

Femtosecond laser has advanced biological researches by providing efficient multiphoton excitation, while maintaining good biological safety (Smith et al., 2005, 2008; Iwanaga, et al., 2006; Liu, X. et al., 2009; Zhao et al., 2009; Wang et al., 2014). Specifically, a transient tightly-focused femtosecond laser irradiation to a localized subcellular structure has been demonstrated to be a highly efficient optical method to directly induce molecular signaling events in single cells (He et al., 2012; Wang et al., 2014, 2018; Cheng et al., 2021). In this protocol, we present a noninvasive ultra-precise laser stimulation (UPLaS) technology to individual mitochondria that excites mitoCa²⁺ oscillations, with little perturbation to MMP or mitoROS. This UPLaS technology is achieved by coupling a femtosecond laser to a confocal microscope, to provide single target cells with a short flash photostimulation. The mitoCa²⁺ oscillation induced by UPLaS initiates the PINK1/Parkin pathway for mitophagy.

The UPLaS technology provides a noninvasive method for simultaneous photostimulation and confocal microscopy of subcellular organelles at a resolution of 628.3 nm. This method is highly flexible, as it can be demonstrated on any two-photon microscope system, and any subcellular organelle can be stimulated by this system.