# Microwave-assisted synthesis of new triangulenium dyes for lifetime imaging microscopy (FLIM)

## TL;DR

A new microwave method creates triangulenium dyes with long fluorescence lifetimes, useful for advanced cell imaging techniques like FLIM.

## Contribution

A novel microwave-assisted synthesis method for triangulenium dyes with improved efficiency and broader applicability.

## Key findings

- ADOTA derivatives accumulate in mitochondria and show short fluorescence lifetimes due to electron transfer.
- DAOTA dyes stain nuclei and endocytic vesicles, with lifetime reductions due to partial aggregation.
- The dyes enable subcellular compartment discrimination via FLIM lifetime contrast.

## Abstract

We introduce a novel microwave-assisted method for the rapid and efficient synthesis of triangulenium dyes, enabling the creation of a broader range of these fluorophores, which are characterized by very long fluorescence lifetimes. We prepared different azadioxatriangulenium (ADOTA) and diazaoxatriangulenium (DAOTA) derivatives and thoroughly characterized their photophysical properties. We investigated their solvatochromic properties and the effects of the solvent, aided by computational chemistry results. The new ADOTA and DAOTA dyes were applied in multiple in cellulo microscopy modalities, including confocal, fluorescence lifetime imaging microscopy (FLIM), and super-resolution imaging. ADOTA derivatives predominantly accumulated in mitochondria, while DAOTAs showed pronounced nuclear staining and participation in endocytic pathways. Notably, these dyes enable clear discrimination of subcellular compartments via lifetime contrast in FLIM: mitochondria, where ADOTAs undergo electron transfer-driven quenching and display short lifetimes; nuclei and DNA, where lifetimes remain long; and endocytic vesicles, where partial dye aggregation leads to lifetime reduction.

The online version contains supplementary material available at 10.1038/s41598-025-31688-6.