RESUMEN
3D super-resolution microscopy based on the direct stochastic optical reconstruction microscopy (dSTORM) with primary Alexa-Fluor-647-conjugated antibodies is a powerful method for accessing changes of objects that could be normally resolved only by electron microscopy. Despite the fact that mitochondrial cristae yet to become resolved, we have indicated changes in cristae width and/or morphology by dSTORM of ATP-synthase F1 subunit α (F1α). Obtained 3D images were analyzed with the help of Ripley's K-function modeling spatial patterns or transferring them into distance distribution function. Resulting histograms of distances frequency distribution provide most frequent distances (MFD) between the localized single antibody molecules. In fasting state of model pancreatic ß-cells, INS-1E, MFD between F1α were ~80â¯nm at 0 and 3â¯mM glucose, whereas decreased to 61â¯nm and 57â¯nm upon glucose-stimulated insulin secretion (GSIS) at 11â¯mM and 20â¯mM glucose, respectively. Shorter F1α interdistances reflected cristae width decrease upon GSIS, since such repositioning of F1α correlated to average 20â¯nm and 15â¯nm cristae width at 0 and 3â¯mM glucose, and 9â¯nm or 8â¯nm after higher glucose simulating GSIS (11, 20â¯mM glucose, respectively). Also, submitochondrial entities such as nucleoids of mtDNA were resolved e.g. after bromo-deoxyuridine (BrDU) pretreatment using anti-BrDU dSTORM. MFD in distances distribution histograms reflected an average nucleoid diameter (<100â¯nm) and average distances between nucleoids (~1000â¯nm). Double channel PALM/dSTORM with Eos-lactamase-ß plus anti-TFAM dSTORM confirmed the latter average inter-nucleoid distance. In conclusion, 3D single molecule (dSTORM) microscopy is a reasonable tool for studying mitochondrion.