Practical considerations in acquiring biological signals from confocal microscope. II. Laser-induced rise of fluorescence and effect of agonist droplet application.

Confocal laser scanning microscopy (CLSM) is extensively used in the study of cellular activities through monitoring the temporal and spatial changes of biologically active molecules such as cAMP and Ca2+ which have been rendered visible by fluorescent labels. During our work with fluo-3 and Ca2+, we noticed two potential sources of artifacts which can make interpretation of the experimental observations difficult. Firstly, the excitation laser light generates heat that enhances the conversion of residual non-fluorescent acetoxymethyl (AM)-esterified indicator to the fluorescent form, thus giving rise to erroneous signals. Secondly, addition of reagents onto the coverslips alters the position of the focal plane, again causing error. In this paper, we present the phenomena and suggest ways to control and eliminate false images.