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

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 power- and 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.


Two-color confocal imaging. Live HeLa cells were stained in DMEM+ containing 2 μM
Lipi-QA and 1% DMSO for 2 h in a CO2 incubator. After washing with fresh medium, the cells were further stained in DMEM+ containing 50 nM tetramethylrhodamine methyl ester (TMRM) and 1% DMSO for 20 min in a CO2 incubator. Then, the cells were washed three times with fresh medium to remove the free probes, and kept in HBSS for confocal imaging. Lipi-QA and TMRM S5 were excited at 488 and 560 nm and detected for 500-550 nm (tg = 6-12 ns) and 570-640 nm (tg = 0-12 ns), respectively, in a line-by-line sequential scan mode.
Statistical analysis. All data were expressed as mean ± S.D., and differences between groups were analyzed by one-way analysis of variance (ANOVA). Tukey's post hoc test was performed by data statistical analysis software (SPSS Statistics, Version number: 18.0.0.282). p < 0.05 was considered as statistically significant, expressing as one star, p < 0.01 as two stars, and p < 0.001 as three stars.

Supplementary chemical synthesis
General. 1 H and 13 C NMR spectra were recorded with a Zhongke-Niujin AS 400 spectrometer (400 MHz for 1 H and 101 MHz for 13 C) in CDCl3 or DMSO-d6. The chemical shifts in 1 H NMR and 13 C NMR spectra were reported in δ ppm using tetramethylsilane as an internal standard. Mass spectra was recorded on a Thermo Fisher ITQ1100 GC/MS mass spectrometer. All reactions were performed under a N2 atmosphere, unless otherwise stated. Commercially available solvents and reagents were used without further purification unless otherwise mentioned.   (100 mL) and acetic acid (100 mL) were successively added to the reaction system and the resulted mixture was further refluxed for 3 h under air condition. After cooling to room temperature, the mixture was filtrated. The precipitated solids were washed with methanol for three times and dried to obtain 7.97 g (18.8 mmol, ~100%) compound 6 as dark red powders. 1 Figure S1 to S17 Figure S1. Top view and side view of the X-ray single crystal structure of Lipi-QA.      S21 Figure S17. The STED laser intensity-dependent resolution and the gating detection delay timedependent resolution. The signal intensity profiles crossed the LDs (shown in the insets of Figure   4A and Figure 4C) were fitted with Gaussian fitting (gray lines).

Discussion of the resolution
Relationship between the Isat value and the resolution. According to the STED super-resolution imaging theory, the resolution Δr is determined by the following equation in principle: where λex is the excitation wavelength, NA is the numerical aperture, ISTED is the intensity of STED laser, and the Isat is the saturation intensity. In general, the λex and NA are constants for a STED super-resolution microscopy. Therefore, decreasing the Isat value is highly desired to achieve high resolution.
Relationship between the Isat value and the fluorescence lifetime. According to the STED super-resolution imaging theory, the saturation intensity Isat is determined by the following equation: where h is the Planck constant, c is the speed of light, σ is the cross section at the STED laser wavelength, τ is the fluorescence lifetime of molecule, λSTED is the wavelength of STED laser.
Since the h, c, and λSTED are constants, increasing the τ value could directly decrease the Isat value and thus contribute to high resolution.

Captions for Movies S1 to S2
Movie S1. Time-lapse STED super-resolution imaging up to 500 STED frames Movie S2. Time-lapse STED super-resolution imaging of a LD fission