Localization of alpha-sarcoglycan and f-actin in human skeletal muscle by fluorescence near field optical microscopy

The alpha-Sarcoglycan and the f-actin are two protein systems with structural and signaling functions, allowing interaction between muscle fibers and extra cellular matrix. Although numerous studies have been conducted on these systems, their localization and distribution patterns along the nonjunctional sarcolemma are not clear. In this paper we first apply fluorescence Near-Field Optical Microscopy to study this problem on human muscle cells, taking advantage of the enhanced spatial resolution of this technique. Samples of human muscle (vastus lateralis) are analyzed, obtained during orthopedic surgery from subjects not affected by neuromuscular diseases. Following the protocol used to carry out indirect immunofluorescence, TRITC-conjugated IgG anti-mouse in goat has been used as the first fluorochrome and, after saturation of residual free binding sites, sections have been incubated with a second antibody conjugated with FITC fluorochrome. Samples, first studied by fluorescence confocal microscopy, show that all tested proteins have a localized, periodic (costameric) spatial distribution. Subsequent SNOM studies have been aimed both to the single localization of the proteins along the cell membrane, and to the double localization of the proteins with respect to each other. The typical costameric band structure has been evidenced in both the fluorescence and the topography maps of dry cells, with a period of similar to 2 mu m for both proteins. A careful correlation between the topography (not available in confocal microscopy) and the fluorescence information permits to draw conclusions on the spatial localization of the two proteins, confirming the results found in confocal microscopy.