Two-photon fluorescent probes for detecting the viscosity of lipid droplets and its application in living cells

Lipid droplets (LDs) are storage organelles at the centre of lipid and energy homeostasis, which act as vital hubs of cellular metabolism and the key to maintaining lipid and energy homeostasis. We synthesized a new two-photon fluorescent probe (CIV) that could detect the viscosity of lipid droplets. The probe is constructed via the typical ICT system of D–π–A using carbazole as the donor and imidazole as the acceptor. With the increase in viscosity from PBS to 99% glycerol, the fluorescence intensity of CIV increased by 13-fold, showing sensitivity and specificity towards viscosity. In addition, CIV showed low toxicity and excellent biocompatibility in cytotoxicity tests, and was successfully used for living cell LD imaging. Taken together, the results widen the way for the development of novel fluorescent probe-based the visualization LDs and detection in solutions, physiology and pathology.


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Characterization 1 H and 13 C NMR spectra were measured on an Avance III 400MHz spectrometer (Bruck Byerspin, Switzerland) in DMSO-d 6 . High-resolution mass spectra (HRMS) were measured on an Apex-Ultra Bruke instrument. The UV-visible-near-IR absorption spectra of dilute solutions were recorded on a U2910 spectrophotometer using a quartz cuvette having 1 cm path length. One-photon fluorescence spectra of dilute solutions were obtained on a HITACH F-2700 spectrofluorimeter equipped with a 450-W Xe lamp. Confocal fluorescence images were obtained with a Nikon A1R confocal laser scanning microscope. To acquire the fluorescence images, TLC analysis was performed on silica gel plates and column chromatography was

Synthesis
Scheme S1 Synthetic route of CIV.
The test results are shown in Figure 1a.

A stock solution (1 mM) of sodium salt ( CNS -、F
inorganic matter (H 2 O 2 ), Organic salt (Acetic acid) and amino acid (Cys、GSH) was prepared in deionized water.

Cytotoxicity assay
The cytotoxicities of the probe CIV to Hela cells was studied by standard MTT assays. 2 × 10 4 cells mL -1 cells were seeded in 96-well plates and then incubated with various concentrations of the probe (0, 2, 5, 10, 20, 30, 50 μM) for 24 h. After that, 10 μL MTT (5 mg·mL -1 ) was added to each well and incubated for another 4 h. Finally, the media was discharged, and 100 μL of DMSO was loaded to dissolve the formazan crystals. The plate was shaken for about 10 min, and each well was analyzed by the microplate reader and detected at the absorbance of 490 nm. The cell viability (%) = (OD sample -OD blank ) / (OD control -OD blank ) × 100 %) .

Fluorescence imaging in cells
HeLa cells of appropriate density were cultured in 90% Dulbecco's Modified Eagle Medium (DMEM, Gibico) supplemented with 10% FBS (Hyclone) and 1% antibiotics (100 U / mL penicillin and 100 μg /mL streptomycin, Hyclone) in an atmosphere of 37 °C and 5% CO 2 . The first group was added with 10 μM of fluorescent probe and cultured for 30 minutes. The second group was added with 10 μM viscosity stimulator Monensin and cultured for 30 minutes. Then the cells were incubated with the probe for additional 30 min. The third group was added 10 μM viscosity stimulating substance Nystatin and cultured for 30 minutes. Then the cells were incubated with the probe for additional 30 min. After washed with PBS for three times, the fluorescence imagings were carried out by a Nikon A1MP inverted fluorescence confocal microscope. The fluorescence emission of the probe was collected at FITC channel (500nm-550nm), the excitation wavelength of one photon imaging was 405 nm and Two photon imaging was 820 nm. Monensin and Nystatin could induce structural changes or swelling, leading to viscosity changes in the lipid droplets.  Figure S1 1 H NMR spectrum of CIV in DMSO-d 6 S10 Figure