Application of an image analyzer to gold labeling in immunoelectron microscopy to achieve better demonstration and quantitative analysis.
An image analyzer was applied to pre- and post-embedding immunogold labeling with 5-nm gold probes in electron micrographs of several skin basement membrane antigens to improve the visualization of immunolabeling. With a TV camera connected to a color image analyzer, an image of an original immunoelectron micrograph was projected on a TV screen. The image was recorded in the analyzer as Record 1. After the floating threshold method procedure to reduce the contrast of the skin structure, electron-dense 5-nm gold particles could be easily detected. With the analyzer, these particles were then suitably enhanced in color and in size and their image was recorded as Record 2. Records 1 and 2 were then overlapped on the TV screen to build up a double-image picture. Compared with the small, electron-dense 5-nm gold particles in the original electron micrograph, ultrastructural localization of bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and the collagenous part of Type VII collagen were more clearly and easily seen, even on low-magnification electron micrographs. The level of background labeling could also be accurately and objectively evaluated. By counting the number of gold particles labeling a certain area and using the analyzer to interpret the result as a diagram, quantitative analysis was also possible. We believe that this technique should be widely applicable to immunogold electron microscopy, not only of skin but also of other substrates of interest.