Correlative microscopy and electron tomography of GFP through photooxidation.
We have developed a simple correlative photooxidation method that allows for the direct ultrastructural visualization of the green fluorescent protein (GFP) upon illumination. The method, termed GRAB for GFP recognition after bleaching, uses oxygen radicals generated during the GFP bleaching process to photooxidize 3,3'-diaminobenzidine (DAB) into an electron-dense precipitate that can be visualized by routine electron microscopy and electron tomography. The amount of DAB product produced by the GRAB method appears to be linear with the initial fluorescence, and the resulting images are of sufficient quality to reveal detailed spatial information. This is exemplified by the observed intra-Golgi stack and intracisternal distribution of a human Golgi resident glycosylation enzyme, N-acetylgalactosaminyltransferase-2 fused either to enhanced GFP or CFP.
Grabenbauer
Markus
M
Cell Biology and Cell Biophysics Program, EMBL, Meyerhofstr. 1, D-69117 Heidelberg, Germany.
Geerts
Willie J C
WJ
Fernadez-Rodriguez
Julia
J
Hoenger
Andreas
A
Koster
Abraham J
AJ
Nilsson
Tommy
T
D015100:3,3'-Diaminobenzidine / Q000737:chemistry; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008854:Microscopy, Electron / Q000379:methods*; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis / Q000378:metabolism; D010084:Oxidation-Reduction; D038761:Photobleaching; D014057:Tomography, X-Ray Computed / Q000379:methods*
D049452:Green Fluorescent Proteins; D015100:3,3'-Diaminobenzidine; D017350:N-Acetylgalactosaminyltransferases