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WI 53706. Colloidal Gold Labeling for Correlative LM, SEM, and TEM Studies. Gold colloids of various sizes can be readily synthesized and conjugated to a variety of molecules, generally without loss of antigenicity or activity of the conjugated molecules. Very small, 1-5 nm, gold particles can be conjugated to small molecules or their active fragments, for example, the Fab portion of IgG, to produce small overall probe sizes permitting a high degree of spatial resolution. If molccule or fragment size is matched with the appropriate gold particle sir.c a 1 to 1 molccule/gold ratio is possible such that semi-quantitative studies are possible, although a number of other factors must also be considered relative to attempts at quantification. These smaller particles can be viewed by high resolution, (field emission) scanning clcctron microscopy. They are readily apparent on uncoated or lhinly coated (1 nm) specimens using beam voltagcs (V,) of 2.5 kV and above in the secondary clcctron (SE) mode. With conventional instrumentation requiring higher kV and thicker coatings 2 contrast is not apparent in the SE mode; however, larger particles can be detected in the SE mode due to their regular round shape. Detectors capable of collecting lower energy backscattered electrons (BSE) also permit unambiguous detection and high resolution imaging of gold particles at lower, 3 to 10 kV, V, . With more conventional BSE detectors higher V, and larger, 7-30 nm, gold particles are required. (Simmons, S.R. et al. 1990. J. Histochem. Cytochem. 38:1781). The dense gold panicles are readily visualized by E M , IVEM, and HVEM at a wide range of V,, 50 kV to loo0 kV. Gold particles can also be identified by their round, regular shape in atomic force microscopy (AFM) with a high degree of spatial resolution. (Simmons, S.R. et al. 1993. Proc. of 51st Microsc. SOC. of Am., G.W. Bailey and C.L. Rieder, eds. pp 230.) Finally, colloidal gold can be visualized in light microscopy (LM) on liviy or otherwise prepared material. Densely labeled arcas appear dark or reddish depending on LM mode and particle size. Individual particlcs, 10 nm and above, which are below the LM resolution limits can be detected via their inflated diffraction image in interference based LM. Suitably prepared material can thus be labeled while living and the distribution and movement or individual panicles followed by video-enhanced LM or possibly by AFM. The exact same particles can later be viewed relative to surface structure by SEM or to internal structure by TEM or HVEM. (Albrecht, R.M. et al. 1993. in Immunocytochcmisuy: A Praclical Approach. J. Beesley (ed). Oxford Press. p 151.
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