Microtomography from limited projections in conventional TEM for 3D reconstruction of an intact cell.
It is possible to generate three-dimensional reconstructions of whole, non-sectioned biological cells in conventional TEM using an 80 kV tungsten source. A TEM specimen stage was modified to accommodate a precise single-axis tilting mechanism controlled by a digital stepping motor interfaced to a computer. For image collection, a video camera was optically coupled to the TEM phosphorescent screen, and the video image was digitized by a frame buffer interfaced to a computer. Specimen tilt and projection image collection were fully computer-automated. This microtomography system design could be readily adapted for most TEMs. Image reconstruction was achieved through computation on projection images from limited tilts; typically less than thirty projection images were needed for a coarse 3D reconstruction. The iterative reconstruction algorithm used certain statistical assumptions about the distribution of image gray values. Since microtomography was performed on non-sectioned whole mount cells viewed under an 80 kV electron beam, methods of embedment-free specimen preparation with chemical fixation and extraction were employed. These methods were utilized successfully to permit good image formation of the entire cell mitotic nucleus a few micrometers in thickness. The 3D reconstruction of a single kidney cell mitotic nucleus was carried out and shown to produce a reasonable microtomogram of gross features like the condensed chromosomes.
Nelson
A C
AC
Bioengineering, University of Washington, Seattle 98195.
Hobbib
W M
WM
D000465:Algorithms; D000705:Anaphase; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure*; D002875:Chromosomes / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D012984:Software; D014054:Tomography / Q000379:methods*