Whole-mount immunoelectron tomography of chromosomes and cells.
Standard immunogold-labeling methods in transmission electron microscopy (TEM) are unable to locate immunogold particles in the depth direction. This inability does not only concern bulky whole mounts, but also sections. A partial solution to the problem is stereo inspection. However, three-dimensional reconstruction of immunogold-labeled structures, that is, immuno-electron tomography (IET), is a correct solution for this inconsistency. Striking improvement in resolution is achieved: the 1.4-nm immunogold particles are shown in IET that are not detected in the original tilt series. IET is not restricted to laboratories with advanced medium- or high-voltage TEM and super-computing facilities; the methods we have developed for whole-mounted chromosomes and also for whole-mounted cytoskeleton of fibroblasts work remarkably well with ordinary 80-kV TEMs equipped with a goniometer to collect tilt series for IET on film. In addition, free programs are available to produce three-dimensional reconstructions even without high-performance computers. These improvements make it possible to many laboratories without modern facilities to perform IET reconstruction with standard TEM apparatus.
Engelhardt
Peter
P
Haartman Institute, Department of Pathology and Virology, Universityof Helsinki, Finland.
Meriläinen
Jari
J
Zhao
Fang
F
Uchiyama
Susumu
S
Fukui
Kiichi
K
Lehto
Veli-Pekka
VP
D000818:Animals; D002460:Cell Line; D002477:Cells / Q000648:ultrastructure; D002642:Chick Embryo; D002875:Chromosomes / Q000648:ultrastructure*; D002877:Chromosomes, Human / Q000648:ultrastructure; D017979:Gold Colloid; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D014054:Tomography / Q000379:methods
D017979:Gold Colloid