SEER of di-8-ANEPPS fluorescence. (A) Structure of di-8-ANEPPS. (B) Its excitation (orange) and emission spectra. Solid black line, emission spectrum measured in mouse muscle (DiFranco et al., 2005). Solid orange line, excitation spectrum, provided by the manufacturer. Broken lines, estimates by J. Vergara and M. DiFranco (personal communication) of excitation and emission spectra, as shifted upon a depolarization of 100 mV. The diagrams at the top indicate excitation and emission lights in two implementations: in a SP2 AOBS conventional pinhole scanner and in the 5 LIVE slit scanner. Lines labeled Ex1 and Ex2 mark the wavelengths used for excitation: 458 and 514 nm in the SP2, and 405 and 488 nm in the 5 LIVE. Rectangles labeled Em1 and Em2 mark the two wavelength ranges where emitted light was collected (524–580 nm and 602–707 nm for the SP2; 505–565 nm and 580–750 nm for the 5 LIVE). Although the use of diffraction gratings and spectral slits in the SP2 assures sharply defined ranges, the definition in the 5 LIVE, which relies on a limited set of fixed optical elements, is less precise (the purple area in the diagram marks a band of residual overlap). Excitation wavelengths and emission ranges were combined as described in the text to obtain F₁₁, F₁₂, F₂₁, and F₂₂. (C and D) Images of fluorescence F₁₁(x,y) and F₂₂(x,y) in an FDB muscle fiber stained with di-8-ANEPPS, obtained using a small pinhole (1.5 Airy diameters). Dynamic studies required line scan images, which were obtained by scanning along the cell surface, as in line a-a of C, using a larger pinhole. ID: 030612a_004. (E) Diagrammatic representation of the membrane systems. Di-8-ANEPPS, present in the extracellular space (orange), will initially stain the plasmalemma and its invaginations, the transverse tubular system. Eventually it will be internalized and also stain the sarcoplasmic reticulum, plus other organelles not shown. Modified from Peachey (1965), with generous help from C.F. Armstrong (University of Pennsylvania, Philadelphia, PA).