E Microscopy

Thick sections are examined with a light microscope, while advanced imaging is done with a research microscope with a digital camera, hooked to a computer for image processing and editing. We use Leica QWin, Nikon or Olympus digital microscopes. Alternatively, the sections can be photographed on film and printed or mounted on 35 mm slides. The resolution of photographic images is superior to that of the digital images and they could be scanned on to a computer for editing. Thin sections are examined by TEM. We use Jeol, Hitachi or Philips microscopes but others such as Zeiss are equally good. The transmission electron microscope has to be maintained, and operated by an experienced technician. Lower magnifications (x 2,000 to x 5,000) are more useful to image whole cells, while

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Figures 5-8 Electron micrographs of thin Araldite sections (~70 nm) stained with uranyl acetate and lead citrate (TEM). (5) Two undifferentiated human ES cells in an EB showing the typical structure. The cells have large nuclei with scanty cytoplasm. Note primitive desmosome (D) between the cells (original magnification: x 42,500) (Reproduced with permission from Sathananthan, 2003).

(6) Cardiac muscle (CM) in a spontaneously differentiating ES cell colony Note the branching fiber and fine microfilaments within the cell (original magnification: x 12,750) (Reproduced with permission from Sathananthan, 2003).

(7) Differentiating ES cell at metaphase of mitosis, showing chromosomes at its equator and a centriole at one spindle pole (original magnification: x 8,750) (Reproduced with permission from Sathananthan et al., 2002).

(8) ES cell colony that has spontaneously differentiated showing goblet-like cells with clear secretions. All cells are probably endodermal in origin (original magnification: x 3,500) (Reproduced with permission from Sathananthan et al., 2002).

high magnifications (x 10,000 to x 100,000) help identify minute cell structures like ribosomes, centrosomes, filaments and membranes in cells (Figures 5-8). Higher contrast could be obtained with lower voltages of 60 or 80 kV and by using smaller apertures in the TEM. Images are photographed on plate film or 35 mm film or saved on computer.

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