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.
Haartman Institute, Department of Pathology and Virology, Universityof Helsinki, Finland.
Peter
P
Engelhardt
Jari
J
Meriläinen
Fang
F
Zhao
Susumu
S
Uchiyama
Kiichi
K
Fukui
Veli-Pekka
VP
Lehto
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