Weakly basic fluorescent dye for vital staining of calcium structures in living organisms

Abstract

Vital dyes capable of staining developing skeletal elements made of carbonate or calcium phosphate contain carboxyl groups that can interact with calcium ions. These dyes are in demand both in research of the biomineralization mechanisms and in the tagging of fish and mollusks in ecological and aquaculture experiments. At the same time, negatively charged dye molecules such as calcein do not penetrate cell membranes well. This necessitates the use of high concentrations, which can cause toxic effects and non-specific staining. This article describes an effective method for synthesizing a new fluorescent dye, QE2, containing a weakly basic amino group and two carboxyl groups in the form of methyl esters capable of hydrolysis in a weakly alkaline environment or under the action of enzymes. The fluorescence of QE2 and its hydrolysis product significantly depends on the polarity of the environment, intensifying in a nonpolar environment with a simultaneous shift in emission from the green-blue to the blue region. The dye is able to easily penetrate living cells, as shown by the example of dinoflagellate culture. Growing organisms with calcium skeletons (gastropods, fish) in the presence of QE2 leads to fluorescent staining of growth areas (gastropod shell aperture, fish spine and fin elements). The QE2 dye can be used to track areas of calcium mineralization, assess the polarity of cell organelles, introduce markers into fish for experiments in ecology and aquaculture, and stain cell cultures and skeletal elements for confocal microscopy studies.