Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a single molecule localization microscopy (SMLM) approach, allowing to reach precision localization around 20 nm, thus providing a 10 time magnification compared to the classical conventional microscopy approach. However, accessing the world of SMLM requires a big step forward and is still paved with many challenges. Our goal was to democratize dSTORM, by developing solutions for a better and easier way to perform nanoscopy imaging. One challenge is the short life of aqueous blinking buffer required to discriminate individual fluorescent molecules. To tackle this, we developed a new buffer, called Eternity Plus, allowing imaging for several weeks and three colors. To evaluate buffer capabilities independently of biological variability, we developed 1 µm calibrated spheres, coated at their periphery with far red blinking fluorophores, known as FluoRef. Imaging deep inside thick samples is challenging for many biological research fields. We propose to challenge an SMLM microscope with non-TIRF illumination and high laser power to image tubulin and myofiber organelles, deep inside isolated muscular fibers. Fine delineation of these structures in control conditions will allow a better comprehension of default structuration in centro-nucleated myopathies. Our workshop will be focused on our recent breakthrough to expand the multicolor capabilities of Eternity-PLUS buffer: a new version applicable to the majority of green emitting fluorochromes opening the way to 3 color dSTORM nanoscopy. Our workshop will be divided into three successive steps: 1- Demonstrate the superior blinking capabilities of our most recent Eternity-Plus buffer in comparaison to Glox buffer using 1 µm calibrated spheres coated with a green fluorophore 2- 3 color Deep SMLM imaging using Eternity-Plus buffer in isolated muscular fibers immuno-detected with microtubules, actinin and mitochondria.