From Live Cells to Caenorhabditis elegans: Selective Staining and Quantification of Lipid Structures Using a Fluorescent Hybrid Benzothiadiazole Derivative

Abstract

The current article describes the synthesis, characterization, and application of a designed hybrid fluorescent BTD–coumarin (2,1,3-benzothiadiazole-coumarin) derivative (named BTD-Lip). The use of BTD-Lip for live-cells staining showed excellent results, and lipid droplets (LDs) could be selectively stained. When compared with the commercially available dye (BODIPY) for LD staining, it was noted that the designed hybrid fluorescence was capable of staining a considerable larger number of LDs in both live and fixed cells (ca. 40% more). The new dye was also tested on live Caenorhabditis elegans (complex model) and showed an impressive selectivity inside the worm, whereas the commercial dye showed no selectivity in the complex model.

Highly selective staining and quantification of intracellular lipid droplets with a compact push–pull fluorophore based on benzothiadiazole

“…CBD-Fluor, however, displayed a stronger bias in this turn-on behavior relative to other LD-specific dyes (Fig. 3B), 13,14,23,26 with a limit of detection in dioxane that was less than 200 pM (Fig. S1 †).…”

“…Notably, CBD-Fluor is also more photostable than other benzothiadiazole dyes reported to stain LDs selectively. 14 CBD-Fluor displays environmentally sensitive emissionhigh emission intensity in non-polar environments and diminished emission in polar environments (Fig. 3A).…”

“…To this end, lipophilic dyes have proven to be invaluable chemical tools for advancing LD research by providing a non-destructive method for the imaging and quantification of LDs in real time-a necessary feature for probing LD dynamics within complex cellular environments. [6][7][8][9][10][11][12][13][14] Of the reported LD stains, Nile Red ( Fig. 1) is most commonly employed as a result of its commercial availability and long history as a reported LD marker.…”

“…These attributes have made benzothiadiazoles attractive for staining lipidic structures [23][24][25] and LDs in particular. 13,14 The benzothiadiazole core can be decorated with targeting agents to help localize the fluorophore to the structure of inter- est. 25 For example, the oleamide in 1 was proposed to assist in cellular uptake and the staining of LDs.…”

“…Indeed, both benzothiadiazole derivatives 3 and 4 have been reported to stain LDs within cells. 13,14 Presumably, this selectivity is due to the fluorescence turn-on behaviour of benzothiadiazoles within the hydrophobic core of lipid droplets. In this regard, the large hydrophobic surface area of 3, 4 and even Nile Red leads to low solubility in water and an increase in non-specific staining of other hydrophobic structures that contribute to background.…”