AIE发光剂在STED显微镜中的应用：低光漂白与高效率

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"低的光漂白和高的发射损耗效率：AIE发光剂作为STED显微镜的荧光探针的潜力" 这篇研究论文探讨了聚集诱导发光（AIE）发光剂在受激发射损耗（STED）显微镜中的应用潜力。STED显微镜是一种超分辨率成像技术，通过抑制荧光区域来实现比传统光学显微镜更高的分辨率。AIE现象是指某些分子在溶液中单体状态时不发光，但在聚集或固态时由于限制的分子运动而表现出强烈的荧光。在传统的荧光探针中，光漂白（光降解导致的荧光减弱或消失）是一个主要问题，影响了长时间观察和多次扫描的可靠性。AIE发光剂因其独特的性质，如低光漂白率，显示出在STED显微镜中作为荧光探针的优势。低光漂白意味着在高能量照射下，这些分子仍能保持稳定的荧光发射，增加了成像的持久性和稳定性。此外，高发射损耗效率是AIE发光剂的另一个关键特性，这使得它们在STED显微镜中特别有用。STED显微镜的工作原理是通过一个高强度的脉冲激光来“关闭”（或损耗）荧光分子的发射，只留下被STED光斑边缘未照射到的分子发光，从而达到亚衍射极限的分辨率。AIE分子的高发射损耗效率意味着它们可以更有效地响应STED激光，进一步提高了图像的分辨率和信噪比。文章中，研究人员Jiaxin Yu、Xianhe Sun、Fuhong Cai、Zhenfeng Zhu、Anjun Qin、Jun Qian、Benzhong Tang和Sailing He分别来自中国浙江大学、华南理工大学-香港科技大学联合研究中心、南华大学发光材料与器件国家重点实验室以及香港科技大学的化学系。他们对AIE发光剂进行了初步研究，结果显示这些分子可能成为STED显微镜的新型高效荧光探针。这篇论文于2015年2月19日提交，4月24日修订，25日接受，并在同年的4月28日发布，最终于5月11日发表。作者之一的Jun Qian是通讯作者，联系邮箱为qianjun@zju.edu.cn。这项工作揭示了AIE发光剂在生物成像、纳米科学和其他需要高分辨率显微镜技术领域中的潜在应用，可能为未来的研究开辟新的方向。

Low photobleaching and high emission depletion

efficiency: the potential of AIE

luminogen as fluorescent probe for STED microscopy

Jiaxin Yu,

Xianhe Sun,

Fuhong Cai,

Zhenfeng Zhu,

Anjun Qin,

Jun Qian,

* Benzhong Tang,

2,3

and Sailing He

State Key Laboratory of Modern Optical Instrumentation, Center for Optical and Electromagnetic Research,

Zhejiang Provincial Key Laboratory for Sensing Technologies, Zhejiang University, Hangzhou 310058, China

SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team, State Key Laboratory of Luminescent

Materials and Devices, South China University of Technology (SCUT), Guangzhou 510640, China

Department of Chemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China

*Corresponding author: qianjun@zju.edu.cn

Received February 19, 2015; revised April 24, 2015; accepted April 25, 2015;

posted April 28, 2015 (Doc. ID 235015); published May 11, 2015

We present a preliminary study which explores the potential of aggregation-induced emission (AIE) luminogen

as a new fluorescent probe for STED microscopy. Compared with Coumarin 102, which is a commonly used

organic fluorophore in STED microscopy, HPS, a typical AIE luminogen, is more resistant to photobleaching.

In addition, HPS-doped nanoparticles have higher emission depletion efficiency than Coumarin 102 in organic

solution. These two advantages of AIE luminogen can facilitate the improvement of spatial resolution, as well

as long-term imaging, in STED microscopy. AIE luminogen will be a promising candidate for STED microscopy

OCIS codes: (160.4890) Organic materials; (160.2540) Fluorescent and luminescent materials; (180.2520)

Fluorescence microscopy; (100.6640) Superresolution.

http://dx.doi.org/10.1364/OL.40.002313

Stimulated emission-depletion (STED) microscopy is a

typical super-resolution imaging technique. It can

achieve very high lateral spatial resolution (<50 nm) and

be used to probe fine biological structure [

1–4]. In STED

microscopy, two laser beams are adopted. One (called

excitation beam) is used to excite the fluorescent probe

and generate emission, and the othe r (called STED

beam) is modulated to the “donut” shape and used to

deplete the emission. The lateral resolution of STED

microscopy relies on the intensity of STED beam [

5].

In order to obtain tens of nanometer-spatial resolution,

very high-power density of STED beam is required.

However, high-power irradiation is harmful to biological

samples. More importantly, fluorescent probes under

high-power excitation suffer a lot from the problem of

photobleaching, which gets the fluorescence signals

weaker and weaker after continuous imaging process.

It also makes STED a nonreversible process, and further

hinders it from achieving high spatial resolution.

Increasing the concentration of fluorescent probe may

be a good way to overcome the problem caused by photo-

bleaching. Unfortunately, most fluorescent probes

adopted in STED microscopy are organic dyes with

aggregation-caused quenching (ACQ) feature [

6], which

means their fluorescence is easily quenched at high

molecule concentrations. Recently, researchers have

proposed some inorganic fluorophores with very little

photonbleaching as replacement [

7–9], which make the

STED microscopy work very well.

In 2001, Luo et al. discovered a phenomenon called

“aggregation-induced emission” (AIE) in a silole fluoro-

gen system [

10], which is opposite to the ACQ effect.

The propeller-shaped AIE molecules are nonemissive

when they are monodispersed in solution, but become

highly fluorescent upon the formation of aggregates. It

provides an ideal solution to the ACQ problem. Based

on this mechanism, AIE dyes with high molecular con-

centration can be designed as nanoparticles, which are

highly emissive, resistant to photobleaching even under

high-power laser irradiation [

11–13], and less toxic to bio-

samples [

11,14,15]. During the past few years, AIE dyes

have been widely applied in bioimaging, e.g., in vitro cell

imaging, macro in vivo imaging, multiphoton micros-

copy, etc. [

15–17]. In this Letter, we studied the potential

of AIE dyes in STED microscopy. Our research was

based on the comparison between hexaphenylsilole

(HPS), a typical AIE luminogen, and Coumarin 102, a

commonly used organic fluorophore in STED micros-

copy [

18]. The experimental results show that HPS

molecules are very resistant to photobleaching in STED

process. In addition, HPS-doped nanoparticles have

higher emission depletion efficiency than Coumarin 102

in organic solution. AIE luminogen will be a good candi-

date for STED microscopy in the future.

The optical setup to perform STED is shown in Fig.

Here we used a 450-nm CW laser as the excitation beam,

and a high-power 532-nm CW laser as the STED beam for

emission depletion. After combined with a dichromic

mirror (DM), these two beams were collimated into an

optical cage system and focused onto the sample via

an excitation objective (100×, NA  1.25, oil immersion;

Olympus). The signals containing fluorescence, stimu-

lated emission, and residual STED light were collected

forward with another objective (10×, NA  0. 25;

Newport). The stimulated emission and residual STED

light (the wavelengths of them were both 532 nm) were

filtered with a 532-nm norch filter. The passed fluores-

cence was coupled into a multi-mode fiber, and finally

detected by a spectrometer (PG2000; Ideaoptics). In

addition, a CCD camera was used to monitor the laser

spots, to ensure the overlap of excitation and STED

beams in STED process.

May 15, 2015 / Vol. 40, No. 10 / OPTICS LETTERS 2313

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