Mitochondria–lysosome interactions are essential for maintaining intracellular homeostasis. Although various fluorescent probes have been developed to visualize such interactions, they remain unable to label mitochondria and lysosomes simultaneously and dynamically track their interaction. Here, we introduce a cell-permeable, biocompatible, viscosity-responsive, small organic molecular probe, Coupa, to monitor the interaction of mitochondria and lysosomes in living cells. Through a functional fluorescence conversion, Coupa can simultaneously label mitochondria with blue fluorescence and lysosomes with red fluorescence, and the correlation between the red–blue fluorescence intensity indicates the progress of mitochondria–lysosome interplay during mitophagy. Moreover, because its fluorescence is sensitive to viscosity, Coupa allowed us to precisely localize sites of mitochondria–lysosome contact and reveal increases in local viscosity on mitochondria associated with mitochondria–lysosome contact. Thus, our probe represents an attractive tool for the localization and dynamic tracking of functional mitochondria–lysosome interactions in living cells.

Neuroregeneration is a dynamic process synergizing the functional outcomes of multiple signaling circuits. Channelrhodopsin-based optogenetics shows the feasibility of stimulating neural repair but does not pin down specific signaling cascades. Here, we utilized optogenetic systems, optoRaf and optoAKT, to delineate the contribution of the ERK and AKT signaling pathways to neuroregeneration in live Drosophila larvae. We showed that optoRaf or optoAKT activation not only enhanced axon regeneration in both regeneration-competent and -incompetent sensory neurons in the peripheral nervous system but also allowed temporal tuning and proper guidance of axon regrowth. Furthermore, optoRaf and optoAKT differ in their signaling kinetics during regeneration, showing a gated versus graded response, respectively. Importantly in the central nervous system, their activation promotes axon regrowth and functional recovery of the thermonociceptive behavior. We conclude that non-neuronal optogenetics targets damaged neurons and signaling subcircuits, providing a novel strategy in the intervention of neural damage with improved precision.

In a transparent medium, lensbased optical microscopy could focus a coherent light beam (e.g., laser) into a tiny spot, whose dimension is comparable to the size of the wavelength of the light. The diameter of the smallest beam waist is about half the size of the wavelength, which is referred to as the diffraction limit. Thus, for visible light, the theoretical diffraction limit is between 200 and 400 nm, much smaller than the size of a single cell (Figure 1A). However, in biological tissues that significantly scatter and absorb visible light, the spatial resolution could be compromised. In multicellular organisms, light absorption limits the penetration depth. The scattering of the light by the opaque biological tissues would expand the volume of light stimulation and reduce the spatial resolution.

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