A new organic molecular probe as a powerful tool for fluorescence imaging and biological study of lipid droplets

Abstract

Background: Lipid droplets (LDs) are critical organelles associated with many physiological processes in eukaryotic cells. To visualize and study LDs, fluorescence imaging techniques including the confocal imaging as well as the emerging super-resolution imaging of stimulated emission depletion (STED), have been regarded as the most useful methods. However, directly limited by the availability of advanced LDs fluorescent probes, the performances of LDs fluorescence imaging are increasingly unsatisfied with respect to the fast research progress of LDs. Methods: We herein newly developed a superior LDs fluorescent probe named Lipi-QA as a powerful tool for LDs fluorescence imaging and biological study. Colocalization imaging of Lipi-QA and LDs fluorescent probe Ph-Red was conducted in four cell lines. The LDs staining selectivity and the photostability of Lipi-QA were also evaluated by comparing with the commercial LDs probe Nile Red. The in-situ fluorescence lifetime of Lipi-QA in LDs was determined by time-gated detection. The cytotoxicity of Lipi-QA was assessed by MTT assay. The STED saturation intensity as well as the powerand gate time-dependent resolution were tested by Leica SP8 STED super-resolution nanoscopy. The time-lapse 3D confocal imaging and time-lapse STED super-resolution imaging were then designed to study the complex physiological functions of LDs. Results: Featuring with the advantages of the super-photostability, high LDs selectivity, long fluorescence lifetime and low STED saturation intensity, the fluorescent probe Lipi-QA was capable of the long-term time-lapse three-dimensional (3D) confocal imaging to in-situ monitor LDs in 3D space and the time-lapse STED super-resolution imaging (up to 500 STED frames) to track the dynamics of LDs with nanoscale resolution (37 nm). Conclusions: Based on the state-of-the-art fluorescence imaging results, some new biological insights into LDs have been successfully provided. For instance, the long-term time-lapse 3D confocal imaging has surely answered an important and controversial question that the number of LDs would significantly decrease rather than increase upon starvation stimulation; the time-lapse STED super-resolution imaging with the highest resolution has impressively uncovered the fission process of nanoscale LDs for the first time; the starvation-induced change of LDs in size and in speed has been further revealed at nanoscale by the STED super-resolution imaging. All of these results not only highlight the utility of the newly developed fluorescent probe but also significantly promote the biological study of LDs.

Solvatochromic Near-Infrared Aggregation-Induced Emission-Active Acrylonitriles by Acceptor Modulation for Low-Power Stimulated Emission Depletion Nanoscopy

“…To the best of our knowledge, this is the lowest I sat value reported for TPA-BT-ANBI in previous research on the STED nanoscopic imaging of LDs using various fluorescent probes (Table S8). − TPA-BT-ANBI exhibited a high depletion efficiency of 91.2% at the depletion laser power of 20.7 mW (Figure B). The low I sat value and high depletion efficiency were beneficial for restricting the background fluorescence and further improving the resolution of TPA-BT-ANBI for STED nanoscopic imaging.…”

Lighting up RNA-specific multi-photon and super-resolution imaging using a novel zinc complex

“…Since Zn-T had good photostability, a large Stokes shift and showed emission at near-infrared wavelengths, we were motivated to use such a complex to image live cell nucleus RNA by stimulated emission depletion (STED) microscopy. 31,32 Fig. 6D shows the nuclear structure under STED and TP confocal microscopy, and the selected areas in the images show nucleoplasmic (Fig.…”

Polarity-Driven Two-Photon Fluorescent Probe for Monitoring the Perturbation in Lipid Droplet Levels during Mitochondrial Dysfunction and Acute Pancreatitis

“…The fluorescence-based molecular probes become emerging tools to provide dynamic information on the local cellular microenvironments such as viscosity, polarity, lipid order, and so forth. − Various strategic fluorescent probes such as rotors, flippers, solvatochromic and push–pull dyes have been employed for detecting LDs in live cells as well as in vivo . Among them, the solvatochromic fluorescent probes show ultrasensitivity toward the order of the LDs and apolar entities. − …”