2.6. Confocal Laser Scanning Microscopy (CLSM) and Image Processing of Fluorescent-Labelled Muscle Sections

For high-resolution microscopy of fluorescent-labelled muscle sections a Leica TCS SP8 confocal laser scanning microscope with acousto-optic tuneable filters, an acousto-optical beam splitter, internal hybrid detectors (HyD SP), and a LMT200 high precision scanning stage was used. Imaging of coverslip-embedded samples was performed via a Leica HC PL APO 63x/1.20 W CORR objective combined with digital zoom factors 1.0 or 2.0. Fluorescence signals were generated via sequential scans, exciting HCS LipidTOX™ Green Neutral Lipid Stain via an argon laser at 488 nm and detecting with an internal HyD at 500–550 nm. AlexaFluor555 counter-stained Spectrin or HCS LipidTOX™ Red Phospholipidosis Detection Reagent were excited by a diode-pumped solid-state laser at 561 nm and detected with internal HyDs at 600–650 nm. The third sequence for visualizing MitoTracker™ Deep Red FM or AlexaFluor647 counter-stained Spectrin involved a 633 nm helium-neon laser for excitation and internal HyD at 650–700 nm for detection. In the last sequential scan DAPI was excited via a 405 nm diode-pumped solid-state laser and detected by an internal HyD at 450–500 nm. Generated images were deconvoluted with Huygens Professional (SVI) and 3D-reconstructed with Imaris software (Bitplane).