Fig. 6. The Golgi apparatus. Analysis for colocalization between CgA fragment-GFP chimeric photoproteins and ß1,4-galactosyltransferase cyan fluorescent protein (GALT-CFP) chimera, a Golgi apparatus marker. (A) 48 hours after transfection with expression plasmids encoding SgP-GFP (a), CgA_1-77-GFP (b), CgA_1-115-GFP (c), CgA_1-224-GFP (d), SgP-CgA-GFP (e), CgAC₁₇E-GFP (f) fusion proteins, together with the expression plasmids encoding for the targeting sequence of ß1,4-galactosyltransferase fused to CFP (GALT-CFP), aldehyde-fixed cells were examined by deconvolution microscopy. GFP was excited at _ex 490±10 nm and imaged at _em 528±38 nm; CFP was excited at _ex 436±10 nm and imaged at _em 465±30 nm. Optical sections along the Z axis were acquired with increments of 0.2 µm using a 100x oil immersion objective (1.4 NA). Data were processed to generate combined 3D/volume views of the GFP and CFP chimera distribution. Representative 0.2 µm XY optical sections acquired in the middle region of the cells, and corresponding three-color mask images generated by Nearcount Image Analyzer are shown. The extent of cyan (CFP) and green (GFP) fluorescent signal 3D colocalization in cells within the boxed areas (crop region) was assessed using Nearcount Image Analyzer software. (B) Nearcount image quantification of GFP (green) chimeras and CFP-Golgi (blue) 3D colocalization. `Overlap' is defined is defined as the total number of blue (CFP) pixels within the crop region that are above a threshold value and have an above-threshold green (GFP) pixel at the same location (same pixel). `Nearness' is defined as the total number of blue (CFP) pixels within the crop region that are above a threshhold value and have an above-threshhold green (GFP) pixel within a 3D 2x2x2 pixel window centered on a green (GFP) pixel. Areas of overlap or nearness are displayed in red (`mask'). Bars, 5 µm.

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