Fig. 1. Subcellular distribution of chimeric photoproteins in PC12 cells. 48 hours after transfection with expression plasmids encoding GFP, SgP-GFP, SgP-CgA-GFP or VAMP2-pHluorin, aldehyde-fixed cells were examined by deconvolution microscopy. Optical sections along the Z axis were acquired with increments of 0.2 µm using 60x or 100x oil immersion objectives (1.4 NA). GFP was excited at _ex 490±10 nm and imaged at _em 528±238 nm. Nuclei were visualized with Hoechst 33342 (_ex 350 nm/_em 461 nm). Data were processed to generate 3D/volume (a1, b1, c1, d1) or section (0.2 µm; a2, b2, c2, d2) views. Bars, 5 µm. Postnuclear supernatants prepared from [³H]-L-norepinephrine-labeled cells transiently expressing GFP (a3), SgP-GFP (b3), SgP-CgA-GFP (c3) or VAMP2-pHluorin (d3), were centrifuged to equilibrium on sucrose density gradients (0.6-2.2 M). Fractions were collected from the bottom of the gradient and assayed for [³H]-L-norepinephrine, sucrose concentration and detection of GFP fluorescence.