Photophysical behaviour and sub-cellular specificity of a pyrene-benzothazolium imaging dye: A study of regio-effect

Pyrene-based small-molecule fluorescent dyes have been widely used in biomedical imaging and sensing applications. Among them, pyrene-based π-acceptor (π-A) type probes are an important class of fluorescent dyes due to their ability to produce a large Stokes' shift (Δλ >100 nm) by strong intramolecular charge transfer (ICT) process. In this work, a pyrene-benzothiazolium-based π-A probe (ETP-2) was synthesized in good yield to study its imaging applications while comparing with previously developed regio-isomer ETP-1. ETP-2 exhibited a large Stokes shift (Δλ ≈100-150 nm) and a bright red-emission (λ_em ≈610-630 nm) due to strong intramolecular charge transfer (ICT) effect. The behaviour of the two regioisomers (ETP-2 and ETP-1) were studied by different photophysical methods including optical spectroscopy, transient absorption (TA) spectroscopy, fluorescence lifetime measurements, and low-temperature fluorescence. Sub-cellular specificity of ETP-2 was studied with several fluorescent markers (mitochondria, lysosome, nucleus, endoplasmic reticulum, and Golgi) and ETP-2 exhibited an exceptional lysosome specificity in A-172 cells (Pearson's correlation coefficient calc. ≈0.9). Fluorescence confocal microscopy imaging and fluorescence spectroscopy-based studies indicated strong evidence to support possible localization of ETP-2 into the membrane regions of the lysosomes.