Quantitative Immunoelectron Microscopy

Abstract

Using antibodies conjugated with colloidal gold particles, immunoelectron microscopy permits the high-resolution detection, localization, and quantification of one or more defined antigens in cellular compartments. These benefits reflect the properties of gold particles (they are electron dense, punctate, and available in different sizes) and the ability of transmission electron microscopy to resolve both particles and compartments. By relating gold marker to cellular fine structure and by taking into account the study design, three pertinent questions can be addressed. When studying a particular group of cells, we might ask: "What is the spatial distribution of gold particles between compartments within a group of cells?" and/or "Is the spatial distribution of gold particles within a group of cells random or due to preferential labeling of compartments?" When comparing two or more groups, a relevant question is: "Are there shifts in compartment labeling distributions in different groups of cells?" Recently, new ways of testing these basic questions have been developed. The efficiency and validity of all these methods rely on sampling, stereologi-cal, and statistical tools. Key processes include random selection of items at each sampling stage (specimen blocks, microscopical fields, etc.), stereological morphometry and/ or unbiased counting, and statistical evaluation of a suitable null hypothesis (no difference in labeling between compartments or groups). This chapter reviews these new methods and illustrates their application with a consistent dataset.