Chem Sci.2017 8(3):1763-1768

Photoactivatable aggregation-induced emission probes for lipid droplets-specific live cell imaging.

Meng Gao,a Huifang Su,b Yuhan Lin,a Xia Ling,a Shiwu Li,a Anjun Qin,*a Ben Zhong Tang*a,b

a Guangdong Innovative Research Team, State Key Laboratory of Luminescent Materials & Devices, South China University of Technology, Guangzhou 510640 China

b Department of Chemistry and Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China.

Correspondence should be addressed to Prof. Anjun Qin (Email: msqinaj@scut.edu.cn) and Prof. Ben Zhong Tang (E.mail: tangbenz@ust.hk)

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Lipid droplets (LDs) as reservoirs of lipids and proteins are dynamic organelles and the elevated expression level of LDs can act as a biomarker of cancer due to the strong requirement of cancer cells for fatty acids and phospholipids.1 To investigate the various biological functions of LDs, it’s highly desirable to realize high spatial and temporal imaging of LDs. Photoactivatable fluorescent probes are potential powerful tools for LDs study through light-controlled high spatiotemporal resolution imaging. There are two major challenges for LDs-specific photoactivatable probes: (1) fluorophores should selectively accumulate in LDs at high concentration to emit bright fluorescence; (2) photoactivatable probes should be easily prepared and specifically accumulate in LDs. However, conventional fluorophores with aggregation-caused quenching (ACQ) drawbacks are difficult to achieve high brightness imaging of LDs.

Aggregation-induced emission (AIE) has recently been proposed as a fundamental solution to solve the fluorescence self-quenching problem.2 AIE-based bioprobes have unique advantages in terms of superior brightness, long-term in situ retention ability, high photostability, and low cytotoxicity. In this study,3 we found that dihydro-2-azafluorenones 1 can efficiently undergo photooxidative dehydrogenation reaction to afford 2-azafluorenones 2 with typical AIE properties (Fig. 1A). With probe 1a as an example, we conducted the photoactivatable imaging experiment for lung cancer HCC827 cells. A very fast light-up process with an excellent photoactivation efficiency was observed via light irradiation at 405 nm with only 1% laser power. Through statistical analyzing the increased fluorescence intensity of 1a in HCC827 cells, an excellent light-up ratio of 265-fold was obtained. Moreover, the co-localization experiment with lipid dye BODIPY493/503 Green showed that the photoactivated 1a-c can specifically accumulate in LDs (Fig. 2).

In contrary with lung cancer HCC827 and A549 cells, only few LDs were observed in normal lung HLF cells by photoactivated 1a (Fig. 3). The much larger number of LDs in lung cancer HCC827 and A549 cells is probably due to their faster growing rate compared with normal lung HLF cells. This experiment suggests that dihydro-2-azafluorenones 1 as photoactivatable and LDs-specific probes can be used to discriminate between cancer and normal cells through their different expression level of LDs.

Light as an external trigger is a valuable and easily controllable tool with high spatial and temporal accuracy. Sequential photoactivation of 1a in selected HCC827 cells can be achieved in a multi-cellular environment (Fig. 4), which suggests that the photoactivatable probes can be used to study LDs’ functions in complex biological environment.

In conclusion, we have developed photoactivatable and LDs-specific probes based on dihydro-2-azafluorenones, which can easily undergo photooxidative dehydrogenation reaction to afford 2-azafluorenones with typical AIE properties. With different amine substituents, dihydro-2-azafluorenones are generally applicable for LDs-specific imaging in live cells with an excellent photo-activation efficiency. They can also be used for sequential photoactivation of selected cells in a multi-cellular environment. Moreover, They can efficiently discriminate between lung cancer and normal lung cells through their different expression levels of LDs. Benefiting from the free of self-quenching in the aggregated state, easy preparation, fast cell uptake, low cytotoxicity, and excellent photoactivation efficiency, the photoactivatable AIE probes developed in this study are expected to have broad applications in biological studies of LDs.

 

 

Fig. 1 (A) Photooxidative dehydrogenation of dihydro-2-azafluorenones 1 to afford 2-azafluorenones 2 with AIE properties. (B) Fluorescence images of live HCC827 cells obtained with increasing irradiation time at 405 nm (1% laser power). (C) Plot of fluorescence enhancement (I/I0) of HCC827 cells with increasing irradiation time.

 

 

Fig. 2 (A, E, I) Bright-field images of HCC827 cells. (B, F, J) Fluorescence images of HCC827 cells from photoactivated 1a, 1b and 1c. (C, G, K) Fluorescence images of HCC827 cells from BODIPY493/503 Green. (D, H, L) The merged images. Scale bar = 20 µm.

 

 

 

Fig. 3 (A) Bright-field image of HCC827 cells. (B) Fluorescence image of HCC827 cells from photoactivated 1a. (C) The merged image. (D) Bright-field image of A549 cells. (E) Fluorescence image of A549 cells from photoactivated 1a. (F) The merged image. (G) Bright-field image of HLF cells. (H) Fluorescence image of HLF cells from photoactivated 1a. (I) The merged image. Scale bar = 20 µm.

 

 

 

Fig. 4 (A) Bright field of HCC827 cells. (B-D) Sequential photoactivation of HCC827 cells (cells 1, 2, and 3) by irradiation at 405 nm (0.2% laser power). Dashed lines indicate the periphery of cells. [1a] = 20 μM. Scale bar = 20 µm.

 

 

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