When cell surface antigens are labeled with the colloidal gold marker, backscattered electron images (BEI) reveal all the gold particles and, therefore, permit total counts. Secondary electron images (SEI) show only a small percentage of the gold particles and are inadequate for quantitative evaluation.
For determination of the cellular labelinq index, a time-consuming method implies the screening of 100 cells by scanninq electron microscopy, at a magnification of approximately 12,000 to 15,000x, with continuous SE/BE shifts. A much more efficient method is to transfer the SEM sample or its equivalent under the light microscope and to count the total number of gold labeled cells in the epi-polarization mode. The total cell count can be evaluated under UV light, taking advantage of the autofluorescence of the glutaraldehyde fixed cells.
de Harven, E. and Soligo, D.
"Immunogold Labeling of Human Leukocytes for Scanning Electron Microscopy and Light Microscopy: Quantitative Aspects of the Methodology,"
Scanning Microscopy: Vol. 1
, Article 27.
Available at: https://digitalcommons.usu.edu/microscopy/vol1/iss2/27