Synthesis and characterization of a new red-emitting Ca2+ indicator, calcium ruby

Area of Science:

• Biochemistry
• Cell Biology
• Fluorescence Imaging

Background:

• Accurate measurement of intracellular calcium ions ([Ca2+]) is crucial for understanding cellular processes.
• Existing calcium indicators may have limitations in brightness, photostability, or spectral properties for specific applications.
• There is a need for novel indicators that are bright, photostable, and suitable for multicolor imaging and challenging cellular environments.

Purpose of the Study:

• To introduce and characterize Calcium Ruby m-Cl, a novel red-emitting calcium indicator dye.
• To evaluate its performance as a tool for measuring intracellular calcium concentration ([Ca2+]).
• To assess its suitability for advanced fluorescence imaging techniques.

Main Methods:

• Synthesis and chemical characterization of Calcium Ruby m-Cl.
• Spectroscopic analysis, including excitation/emission spectra, molar extinction coefficient, and quantum yield determination.
• Evaluation of conjugation capabilities via EDC and click chemistry.
• Assessment of indicator performance in cellular imaging experiments.

Main Results:

• Calcium Ruby m-Cl exhibits red emission (579/598 nm peak excitation/emission) upon visible light excitation.
• The dye possesses a large molar extinction coefficient and high quantum yield, classifying it as a very bright indicator.
• It features a side arm enabling straightforward conjugation using EDC or click chemistry.
• Demonstrated utility for imaging intracellular calcium concentration ([Ca2+]) in multicolor fluorescence applications and in the presence of yellow-green autofluorescence.

Conclusions:

• Calcium Ruby m-Cl is a highly effective red-emitting calcium indicator.
• Its brightness and spectral properties make it a valuable alternative to existing dyes.
• The dye's conjugation flexibility and performance in challenging imaging conditions highlight its potential for advancing cellular calcium research.