Fluoview fv 300 confocal system

Confocal images were acquired on a laser scanning confocal microscope. Excitation was provided by Ar (488 nm), green He-Ne (543 nm), and red He-Ne (633 nm) lasers. Images were collected on two separate confocal systems. Some images were collected on a FluoView FV300 Confocal System (version 4.3, Olympus; Center Valley, PA) interfaced to an Olympus IX-70 inverted microscope. Data were collected using an oil immersion 40X objective in series of optical sections at intervals of 0.35–1 μm, with two- or three-frame Kalman averaging for each section. Other images were collected on a FluoView FV1000 Confocal System (version 1.6, Olympus) interfaced to an Olympus IX-81 inverted microscope. Data were collected with an oil immersion 60X objective in series of optical sections at 0.44-μm intervals, with three-frame Kalman averaging for each section.

An Olympus Fluoview FV 300 confocal system featuring three lasers and an Olympus BX60 fluorescent microscope (Olympus America, Melville, NY) was used to collect images of NMJs. Using a 100x oil immersion objective, it was initially established that the entire NMJ was within the longitudinal borders of the myofiber and that damage to the structure had not occurred during sectioning. A detailed image of the entire NMJ was constructed from a z-series of scans taken at 1 µm thick increments. Digitized, two-dimensional images of NMJs were stored on the system’s hard drive and later quantified with the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). A minimum of 10 NMJs per muscle were quantified and measurements were averaged to represent synaptic morphology within that muscle.
In the quantification of muscle fiber profiles, an Olympus BX41 bright field microscope was used in conjunction with a 40X objective. Myofiber cross-sectional areas were quantified with the Image Pro-Express software. A random sample of 150–200 myofibers from each muscle was analyzed to determine average myofiber size (i.e., cross-sectional area) and fiber type composition (% of total number of fibers examined) for that muscle.

An Olympus Fluoview FV 300 confocal system featuring three lasers and an Olympus BX60 fluorescent microscope (Olympus America, Melville, NY) was used to collect, and store, images of NMJs. Using a 100x oil immersion objective, it was initially established that the entire NMJ was within the longitudinal borders of the myofiber and that damage to the structure had not occurred during sectioning. A detailed image of the entire NMJ was constructed from a z-series of scans taken at 0.5 μm thick increments. Digitized, two-dimensional images of NMJs were stored on the system’s hard drive and later quantified with the Image-Pro Plus software (Media Cybernetics, Silver Spring, MD). In each muscle, 10–12 NMJs were quantified and measurements were averaged to represent NMJ morphology within that muscle.
In the quantification of myofiber profiles, an Olympus BX41 microscope equipped with fluorescence capacity (X-Cite, Excelitas Technologies, Ottawa, Canada) was used in conjunction with Infinity Analyze software (Lumenera Corporation, Ottawa, Canada). A random sample of 125–150 myofibers from each muscle was analyzed to determine average myofiber size (cross-sectional area), fiber type composition (% of each fiber type analyzed for that muscle), as well as percentage of total myofiber area measured that was occupied by each individual fiber type.