Two-photon AIE bio-probe with large Stokes shift for specific imaging of lipid droplets

A novel AIEgen with prominent two-photon excitation was rationally developed for specific lipid-droplet imaging in cells and tissues.

measured by two-photon excitation fluorescence method using rhodamine 6G and fluorescein as references. 1 All the experiments, concerning the usage of fetal bovine serum and fixed mice liver slices, etc. were performed in compliance with the relevant laws and institutional guidelines. The institutional committee has approved the experiments.

Preparation of lipid and glyceryl trioleate solutions
Phosphate buffered solution (PBS)+1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, purchased from Avanti), PBS+DMPC+glyceryl trioleate (TAG, purchased from Sigma): 200 μL of 10mg/mL DMPC chloroform solution and 0 or 4 mg TAG, were added into an empty clean vial and the solvent was then removed under nitrogen. Afterwards, 5 mL of PBS was added and the solution was subjected to sonication for 10 min. 4 μL of 5 mM TPA-BI DMSO solution was added into 1 mL of the following solutions (PBS, PBS+ DMPC, PBS+DMPC+TAG). The resulting solutions were mixed by vortex. The final solution concentration was 20 μM for TPA-BI, 400 μg/mL for DMPC and 800 μg/mL for TAG.

Cell culture
HeLa cells were cultured in MEM containing 10% FBS and antibiotics (100 units per mL penicillin and 100 μg/mL streptomycin) in a 5% CO 2 humidity incubator at 37 °C.

Cell viability
Cells were seeded in 96-well plates at a density of 5000 cells per well. After culture for 24 h, the medium in each well was replaced by 100 μL of fresh medium containing different concentrations (0, 0.5, 1, 2.5, 5, 10 and 20 μM) of TPA-BI. The volume fraction of DMSO was below 0.2%. After 24 h, 10 μL of MTT solution (5 mg/mL in PBS) was added into each well. After 4 h incubation, 100 μL of SDS-HCl aqueous solution (10% SDS and 0.01 M HCl) was added to each well. After incubation for 6 h, the absorption of each well at 595 nm was recorded via a plate reader (Perkin-Elmer Victor3 TM ). Each trial was performed with 6 wells parallel.

Cell treatment with oleic acid
HeLa cells were grown overnight on a 35-mm Petri dish with a cover slip. The cells were incubated with 50 μM of oleic acid for certain time (36 h) to induce lipid droplet formation. 2

Cell imaging
HeLa cells were grown on a cover slip overnight in a 35-mm petri dish. The cells were stained with certain dye at certain concentration for certain time (by adding 2 μL of stock solution in DMSO to a 2 mL of culture medium with DMSO < 0.1 vol %). The cells were imaged under a fluorescent microscope (upright BX41 Microscope) using proper excitation and emission filters for each dye: for TPA-BI, excitation filter = 400440 nm, dichroic mirror = 455 nm, and emission filer = 465 nm long pass; for BODIPY, excitation filter = 460490 nm, dichroic mirror = 505 nm and emission filter = 515 nm long pass.

Photostability
On a confocal microscope (Leica DMI 6000 Fully Motorized Inverted Microscope), the dyes were excited with 442 nm or 488 nm laser light for one-photon imaging and 840 nm for two-photon imaging. Imaging parameters were set for each dye individually to obtain optimal images. Repeated image scans were taken. On each series of scans, five/six regions of interest (ROIs) with several LDs were defined. The first scan of each ROI was set to 100%. Then the pixel intensity values for each ROI were averaged and plotted against the scan number. The resulting curve represented the bleaching rate that an experimentalist would encounter.

Flow cytometry
Six medium plates of HeLa cells were grown overnight. Then the cells were incubated with 50 μM of oleic acid for 0, 2, 3 and 4 h before collection for flow cytometry. The cells were stained with 1 μM of TPA-BI for 10 min and 1 μg/mL BODIPY for 15 min, respectively, and washed with PBS. The cells were analyzed by flow cytometry (Becton Dickinson FACS Aria IIIu). 10000 events were taken for each trial.

Synthesis of TPA-BI
TPA-BI (1) was synthesized following the synthetic route shown in Scheme 1. The key intermediates 2 and 3 were prepared according to Scheme S1 and the procedures described below. Scheme S1 Synthetic route to 1.

Fig. S16
Fluorescence spectra of 20 μM of TPA-BI in PBS, PBS with DMPC, and PBS with DMPC and TAG excited at 840 nm. The spectra were averaged from three measurements using Gaussian single peak fitting. The peak intensity was 6, 15 and 107 arbitrary unit, respectively.