OBSERVATIONS OF THICK AND THIN SECTIONS IN A FIELD EMISSION SCANNING 
ELECTRON MICROSCOPE

William P. Wergin, Stephane Roy, Eric F. Erbe, Charles A. Murphy and Christopher D. Pooley

Electron Microscopy Laboratory; USDA-ARS; Beltsville, MD 20705

Larvae of the nematode, Steinernema carpocapsae Weiser strain All, were cryofixed and freeze-
substituted for 3 days in acetone containing 2% osmium tetroxide according to established procedures1. 
 Following chemical fixation, the nematodes were brought to room temperature, embedded in Spurr's 
medium2 and sectioned for observation with a Hitachi S-4100 field emission scanning electron 
microscope that was equipped with an Oxford CT 1500 Cryotrans System.  Thin sections, about 80 nm 
thick, similar to those generally used in conventional transmission electron microscope (TEM) studies3 
were mounted on copper grids and stained with uranyl acetate for 30 min and lead citrate for 5 min.  
Sections about 2 mm thick were also mounted and stained in a similar fashion.  The grids were mounted 
on an Oxford grid holder, inserted into the microscope and onto a cryostage that was operated at 
ambient temperature.  Thick and thin sections of the larvae were evaluated and photographed in the 
SEM at different accelerating voltages.  Figs. 4 and 5 have undergone contrast conversion so that the 
images would resemble transmitted electron micrographs obtained with a TEM.

Obtaining ultrastructural details of embedded tissue in the SEM is partly a function of section thickness 
versus accelerating current (Figs. 1-3).  At low voltages, the secondary electron signal is emitted mainly 
from the surface of the section; fine structural details are not observed until the beam penetrates the resin 
and excites secondary electrons from the heavy metal stains on the embedded tissue.  Images of thin 
sections, showing well preserved biological structure can be easily observed at 20 kV (Fig. 4).  At 30 
kV the beam of the field emission SEM is also capable of penetrating sections that are 2 mm thick.  As a 
result stereo images, which resemble those taken with a high voltage TEM, can also be recorded (Fig. 
5).

1.  J. C. Gilkey & L. A. Staehelin, J. Electron Microsc. Tech. 3(1986)177.
2.  A. R. Spurr, J. Ultrastruct. Res.  5(1969)31.
3.  W. P. Wergin & and B. Y. Endo, J. Ultrastruct. Res. 56(1976)258.

Figures 1 to 3.  Thin section of nematode larvae illustrating the effects of three different kVs.  At 1 kV 
(Fig.1) the secondary electron signal appears to arise principally from the surface of the section.  At 3 
kV (Fig. 2), the contribution of the surface of section is less; the deeper penetrating beam results in 
greater signal from the heavy metal stains of the sectioned nematode as well as the underlying grid bars. 
 The effect of the heavy metal staining on the secondary electron signal is even greater at 5 kV (Fig. 3) 
where the beam penetrates through the section resulting in an intense signal from the grid bars.  Bar = 
0.1 mm.
Figure 4.  High magnification of a thin section of the nematode taken at 20 kV.  Figure illustrates 
somatic muscle tissue including muscle fibers and mitochondria.  SEM image has undergone contrast 
conversion.  Bar = 0.5 mm.
Figure 5.  Stereo pair of images from section estimated to be greater than 2 mm in thickness recorded at 
30 kV. Figure illustrates the cuticle, hypodermis, somatic muscles and other tissues of the nematode.  
SEM image has undergone contrast conversion.  Bar = 2.0 mm.