Chemiluminescence microscopy single cell imaging using esterase triggered 1,2-dioxetanes.

Area of Science:

• Biomedical Imaging
• Molecular Imaging
• Cellular Imaging

Background:

• Chemiluminescence microscopy (CLm) offers advantages over fluorescence imaging, including reduced autofluorescence and photobleaching.
• Current CLm applications using 1,2-dioxetane probes are limited by challenges in cell uptake, signal intensity, and specialized microscopy equipment.
• Existing bioluminescence microscopy relies on genetically modified organisms, limiting its broad applicability.

Purpose of the Study:

• To develop a novel chemiluminescent 1,2-dioxetane and microscopy system for low-background biological imaging.
• To overcome limitations of existing chemiluminescence microscopy techniques, particularly for imaging intracellular activities in unmodified cells.
• To establish a generalizable framework for diverse clinical sample imaging.

Main Methods:

• Development of a home-built chemiluminescence microscope optimized for imaging 1,2-dioxetanes.
• Synthesis and utilization of acetoxymethyl ester functionalized 1,2-dioxetanes (probes 2 and 3).
• Characterization of probe uptake, retention, and luminescence imaging in unmodified A549 human lung epithelial cells.

Main Results:

• The developed chemiluminescence microscope successfully imaged nanomolar concentrations of luminescent 1,2-dioxetanes.
• Functionalized 1,2-dioxetanes demonstrated efficient uptake and retention in A549 cells.
• Intracellular esterase activity was successfully imaged in A549 cells using the chemiluminescence microscopy system.

Conclusions:

• This study presents a complementary chemiluminescent 1,2-dioxetane probe and microscopy system.
• The developed system provides a generalizable framework for imaging various clinically relevant samples.
• This work advances the field of low-background biological molecular imaging.