Electron tomography of amplified nanogold immunolabelling: Improvement of quality based on alignment of projections with sinograms and use of post-reconstruction deconvolution.
Electron tomography of immunolabelled proteins identified with amplified nanogold particles imaged by Scanning and Transmission Electron Microscopy within thick sections is a powerful method to investigate the three-dimensional organization of complex cellular machineries. In order to increase the overall quality of the reconstructed cube, we have developed two methods that improve the tomographic reconstruction process. We first performed a very precise alignment of the projections before reconstruction with a technique using sinograms. After reconstruction, we propose to compute image restoration by calculating the Point Spread Function of the projection/back-projection system and to use it to deblur the reconstructed cubes. Improvement in the quality of the reconstructed cubes is demonstrated on images of nucleolar proteins tagged with EGFP and immunolabelled with nanogold particles.
Tchelidze
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P
Unité MéDIAN, CNRS UMR 6142, UFR de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France.
Sauvage
C
C
Bonnet
N
N
Kilian
L
L
Beorchia
A
A
O'Donohue
M F
MF
Ploton
D
D
Kaplan
H
H
D006046:Gold / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D007624:KB Cells; D008854:Microscopy, Electron / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods; D049329:Nanostructures / Q000737:chemistry; D009687:Nuclear Proteins / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000235:genetics; D014057:Tomography, X-Ray Computed / Q000379:methods*
D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006046:Gold