Artifacts in Fluorescence Ratio Imaging

Abstract

Successful development of reliable fluorescent ratio indicators for ion concentration, membrane fluidity and membrane potential and steady improvement of the imaging equipment (microscopes, detectors, computers) have made fluorescence ratio imaging microscopy a widely used method in biomedicai research (Slavík, 1994). An increasing number of papers deal especially with Ca²⁺ and pH measurements. The proper interpretation of experimental data requires not only a profound knowledge of the fluorescent probe used and a reliable calibration standard curve but one must be also aware of artifacts inherent in the opto-electronical level of the technique. These artifacts can arise from the following causes: blurring of the image due to the involvement of out-of-focus light, background fluorescence, improper adjustment of the detection part of experimental setup, movement of the sample, and bleaching of the dye.