VALIDATION OF IMMUNOHISTOCHEMICAL QUANTIFICATION IN CONFOCAL SCANNINGLASER MICROSCOPY - A COMPARATIVE-ASSESSMENT OF GAP JUNCTION SIZE WITHCONFOCAL AND ULTRASTRUCTURAL TECHNIQUES

Confocal scanning laser microscopy (CSLM) is increasingly being used t o image antibody-labeled structures visualized with a fluorescent seco ndary antibody. Such digital images are routinely stored on computer a nd are well suited to quantitative analysis. Although theoretical aspe cts of CSLM imaging and resolution are well defined, information is la cking on the relationship observed between measurements of fluorescent antibody-labeled structures and the size of the same structures as de termined by electron microscopy (EM). In the present study we examined this relationship for the cardiac gap junction. Data on the size of i mmunofluorescent-labeled gap junctions were acquired by two methods of analysis from CSLM images and compared statistically with measurement s of gap junction size obtained by freeze-fracture EM. The freeze-frac ture data were compared before and after exclusion of small junctions, corresponding to those that theoretically would not have been detecte d in CSLM analysis. The data obtained by the different methods were si milar but not identical, reflecting the advantages and limitations of each technique. However, the comparison did indicate that with appropr iate sample preparation and orientation, accurate and rapid analysis c an be achieved by CSLM, particularly when digital semi-automated techn iques are employed.