Background

Mitochondria are crucial organelles responsible for ATP production, metabolic regulation, calcium homeostasis, and participation in cellular signaling processes [1–5]. The mitochondrial inner membrane (IM) is a crucial structure within mitochondria, containing the oxidative phosphorylation complexes responsible for ATP synthesis. These complexes on the IM play a key role in cellular energy supply by hosting the entire respiratory chain [6,7]. The IM folds into cristae to increase the surface area, thereby supporting greater ATP production. The shape of the cristae is altered by mitochondrial dynamics, which can affect mitochondrial respiration [8].

The diameter of mitochondria ranges from 200 to 700 nm, with cristae spacing typically around 70 nm [9]. Conventional confocal microscopy has a resolution limited to approximately 200 nm, which is insufficient to resolve the details of these structures; however, the advent of super-resolution microscopy has enabled the visualization of cristae [10]. Stimulated emission depletion (STED) microscopy, in particular, achieves high resolution by employing a doughnut-shaped depletion laser to selectively erase peripheral fluorescence following sample excitation, thereby improving the point spread function (PSF). STED technique provides a spatial resolution of 50 nm and temporal resolution of 1 frame per second, making it a powerful tool for dynamic imaging of mitochondrial cristae at the single-crista level.

In the STED system, the intensity of the depletion beam is several thousand times higher than that of the excitation beam, requiring fluorophores in the fluorescent probes to possess exceptional photostability to avoid quenching. Therefore, we have designed a novel fluorescent dye for STED imaging. HBmito Crimson is characterized by exceptional photostability, targeted accumulation on the mitochondrial IM, and emission only upon binding to the IM, as shown in Figure 1A. In pure organic solvents or water, HBmito Crimson exhibited its absorption peak at 660 nm and an emission peak at 688 nm (Figure 1B). The emission spectrum tail of HBmito Crimson extends toward 775 nm, generating a small peak near this wavelength. This reduces the dye’s saturation power, significantly enhancing the depletion efficiency of the 775 nm laser [11].

Figure 1. Structural and spectral properties of HBmito Crimson for mitochondrial labeling. A. The chemical structure of HBmito Crimson is used for the specific labeling of the mitochondrial inner membrane. B. The absorption and emission spectra of HBmito Crimson, which can be depleted using a 775 nm laser.

In this protocol, we describe in detail the preparation of cells, labeling methods with HBmito Crimson, image acquisition, and image processing methods suitable for confocal and STED microscopy imaging.