Fused Thiophene-S,S-dioxide-Based Super-Photostable Fluorescent Marker for Lipid Droplets

Abstract

Lipid droplets (LDs) are essential organelles in most eukaryotes, and tracking intracellular LD dynamics using synthetic small molecules is crucial for biological studies. However, only a limited number of fluorescent markers that satisfy all requirements, such as the selective staining of LDs, high photostability, and sufficient biocompatibility, have been developed. Herein, we report a series of donor-π-acceptor dyes based on the thiophene-containing fused polycyclic scaffold [1]­benzothieno­[3,2-b]­[1]­benzothiophene (BTBT), in which either or both thiophene rings are oxidized into thiophene-S,S-dioxide to form an electron-accepting building block. Among these dyes, LAQ1 satisfied all the aforementioned requirements and allowed us to capture ultrasmall LDs on the endoplasmic reticulum (ER) membrane by stimulation emission depletion (STED) microscopy with a super-resolution below the diffraction limit of light. Moreover, the extremely high photostability of LAQ1 enabled recording the lipolysis of LDs and the concomitant lipogenesis, as well as long-term trajectory analysis of micro-LDs at the single-particle level in living cells.

Recent advances in fluorescent probes for lipid droplets

“…24), wherein diphenylamine moieties were directly conjugated to the electron acceptor ([1]benzothieno[3,2-b][1] benzothiophene). 58 Among these dyes, 59 showed appropriate lipophilicity and resulted in a high signal-noise ratio for LDs imaging. Therefore, 59 was used to capture the ultrasmall LDs on the ER by STED microscopy.…”

A negative-solvatochromic fluorescent probe for visualizing intracellular distributions of fatty acid metabolites

“…26 and Supplementary Note 1 ). Prior to cell imaging, to evaluate whether AP-C12 was capable of discriminating cell environments based on differences in lipid composition, we measured the excitation and emission spectra of AP-C12 in an aqueous buffer (1% DMSO; pH = 7.4), phospholipid vesicles (large unilamellar vesicles (LUVs) prepared from 1,2-dioleoyl- sn -glycero-3-phosphocholine), and soybean oil (1% CHCl 3 ), which are model systems that mimic the local environments of cytosol and the mitochondrial matrix, phospholipid membranes, and LDs 26 , respectively. The excitation spectra and fluorescence spectra of AP-C12 showed distinct negative solvatochromism, as was observed for AP-Me in different solvents (Fig.…”

STED Nanoscopy Imaging of Cellular Lipid Droplets Employing a Superior Organic Fluorescent Probe

“…The fwhm resolution of the STED image is up to 65 ± 7 nm, which is substantially higher than the confocal image of 250 ± 6 nm (Figure c,d) and belongs to one of the leading results of LDs imaging (Table S1). − Notably, this high-resolution STED image is obtained under a relative weak STED laser intensity (16 MW cm –2 ), which is highly desired for live cell imaging with decreased photodamage. Further enhancing the fwhm resolution better than 65 nm may be realized by STED imaging with time-gated detection.…”

“…In the case of the STED nanoscopy imaging technique, it has two intrinsic requirements for fluorescent probes: (1) the probe should be able to be efficiently depleted by a STED laser; and (2) the probe should display very high photostability because of the tremendous energy of STED laser. , Since the traditional organic fluorescent probes could not satisfy these harsh demands, great efforts have been donated to develop new organic fluorescent probes capable for STED nanoscopy imaging. As a result, a few superior fluorescent probes have been reported very recently for STED nanoscopy imaging of various cellular organelles, e.g., LDs, − mitochondrial membrane, − cytoskeleton, , and so on. − Besides these organic molecular fluorescent probes, some organic/inorganic nanoparticles-based fluorescent probes have also emerged as powerful tools for STED imaging. − These works highlight the advantage of STED nanoscopy imaging over the traditional confocal/two-photon imaging, and significantly promote the relative study of cell biological on the nanoscale. However, fluorescent probes that are competent for STED nanoscopy imaging are quite limited.…”