Synthesis, characterisation and application of red-emitting BODIPY dyes for multiphoton microscopy of lipid droplets

This study introduces two novel red-emissive BODIPY dyes, each functionalized with methoxystyryl groups at the 3- and 5-positions to bathochromically shift spectral bands. Obtained via facile synthetic pathways, the dyes can be excited using red single-photon or near-infrared two-photon excitation. The latter is of key importance, with long-wavelength excitation being advantageous for live-cell microscopy as it offers increased tissue penetration, reduced scattering, and minimal autofluorescence. Comprehensive structural, computational, and photophysical characterisation highlight the suitability of both dyes for use in live-cell laser scanning confocal microscopy (LSCM) of mouse embryonic fibroblast (NIH-3T3) cells. The materials are characterised by high brightness with high molar absorption coefficients and photoluminescence quantum yields. Additionally, two-photon absorption cross-sections were measured as 723 GM and 502 GM (1 GM = 10-50 cm 4 s photon -1 ) at 840 nm, respectively, making the dyes suitable for multiphoton microscopy applications. Dosing protocols were developed for single-photon and multiphoton LSCM, and co-staining confirmed strong localisation within lipid droplets (LDs). LDs are an important organelle to study regarding manifestation of diseases such as diabetes and cancer, as well as monitoring of distribution of lipophilic intracellular material. Cytotoxicity assays at a range of concentrations (500 nM to 5 μM) for up to 24 hours revealed both dyes can be used for prolonged live-cell microscopy without significant alterations to cellular viability. • Novel organic compounds for two photon imaging of lipid droplets in live cells with high two-photon cross sections. • Favourable photophysical characteristics in the red end of the electromagnetic spectrum. • Lipid droplets have been receiving increasing attention due to their role in diseases such as diabetes and cancer but they also play an essential role in energy storage, cell signalling and lipid and protein handling amongst others. • Localization in lipid droplets confirmed by co-localization with commercially available lipid droplet stain.