Uncovering the impact of UV radiation on mitochondria in dermal cells: a STED nanoscopy study.

Mitochondria are essential organelles in cellular energy metabolism and other cellular functions. Mitochondrial dysfunction is closely linked to cellular damage and can potentially contribute to the aging process. The purpose of this study was to investigate the subcellular structure of mitochondria and their activities in various cellular environments using super-resolution stimulated emission depletion (STED) nanoscopy. We examined the morphological dispersion of mitochondria below the diffraction limit in sub-cultured human primary skin fibroblasts and mouse skin tissues. Confocal microscopy provides only the overall morphology of the mitochondrial membrane and an indiscerptible location of nucleoids within the diffraction limit. Conversely, super-resolution STED nanoscopy allowed us to resolve the nanoscale distribution of translocase clusters on the mitochondrial outer membrane and accurately quantify the number of nucleoids per cell in each sample. Comparable results were obtained by analyzing the translocase distribution in the mouse tissues. Furthermore, we precisely and quantitatively analyzed biomolecular distribution in nucleoids, such as the mitochondrial transcription factor A (TFAM), using STED nanoscopy. Our findings highlight the efficacy of super-resolution fluorescence imaging in quantifying aging-related changes on the mitochondrial sub-structure in cells and tissues.

Spatial localization of charged molecules by salt ions in oil-confined water microdroplets.

Cells contain more than 100 mM salt ions that are typically confined to dimensions of 5 to 10 micrometers by a hydrophobic cellular membrane. We found that in aqueous microdroplets having the same size as cells and that are confined in hydrocarbon oil, negatively charged molecules were distributed rather uniformly over the interior of the microdroplet, whereas positively charged molecules were localized at and near the surface. However, the addition of salt (NaCl) to the microdroplet caused all charged molecules to be localized near the oil-water interface. This salt-induced relocalization required less salt concentration in microdroplets compared to bulk water. Moreover, the localization became more prominent as the size of the microdroplet was reduced. The relocatization also critically depended on the type of oil. Our results imply that salt ions and different hydrophobic interfaces together may govern the local distribution of charged biomolecules in confined intracellular environments.

Restricted intramolecular rotation of fluorescent molecular rotors at the periphery of aqueous microdroplets in oil.

Fluorescent molecular rotor dyes, including Cy3, Cy5, and Alexa Fluor 555, dissolved in micron-sized aqueous droplets (microdroplets) in oil were excited, and the fluorescence intensity was recorded as function of time. We observed lengthening of the fluorescence lifetime of these dyes at the water-oil periphery, which extended several microns inward. This behavior shows that intramolecular rotation is restricted at and near the microdroplet interface. Lengthened lifetimes were observed in water microdroplets but not in microdroplets composed of organic solvents. This lifetime change was relatively insensitive to added glycerol up to 60%, suggesting that solution viscosity is not the dominant mechanism. These restricted intramolecular rotations at and near the microdroplet periphery are consistent with the reduced entropy observed in chemical reactions in microdroplets compared to the same reaction conditions in bulk solution and helps us further understand why microdroplet chemistry differs so markedly from bulk-phase chemistry.