Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China
Abstract:Optical microscopy has the advantages of real-time, non-invasive, tomography, three-dimensional imaging and living imaging. However, its spatial resolution cannot exceed half wavelength due to the existence of optical diffraction limit, which limits the development of optical microscopy. The primary task of super-resolution imaging is to break the diffraction limit and improve the resolution of optical microscopy for study of subcellular structure. Many kinds of super-resolution imaging technologies have been reported, among which the stimulated emission depletion (STED) microscopy is the earliest imaging technology to break the optical diffraction limit at present. STED microscopy can achieve nanometer-scale spatial resolution by breaking the optical diffraction limit with pure optical methods and a clever optical design. However, the application of STED microscopy in biomedicine, especially in live cell imaging is limited by high illumination power of STED light. In this paper, a new type of STED probe has been developed. The spectral analysis results show that the peak of the excitation and emission spectrum of this probe is as far as 122 nm away from each other, which is very suitable for the study of STED super-resolution because of its long stokes redshift. After colocalization with commercial mitochondrial dyes, it was found that the probe had a higher localization coefficient with commercial dyes and could be well positioned on mitochondrial organelles. At the same time, it was found that strong mitochondrial signal could be detected with low-power excitation light (only 1 μW in the experiment), and can get higher resolution of 62 nm under the STED light with 39.5 mW. The result of measuring the transverse resolution obtained by STED light under different power shows that the saturated light power of the probe is 3.5 mW (1.1 MW·cm–2). Through the anti-bleaching testing, the probe still has a strong fluorescence intensity after more than 300 times of high power light irradiation, which indicates that the probe has a strong anti-bleaching property. Through a series of tests, this paper present a novel STED probe which has good mitochondrial targeting, excellent photobleaching-resistance, high resolution and low saturation power, which provides a new research tool for long-term live cell mitochondrial super-resolution imaging. Keywords:stimulated emission depletion microscopy/ super-resolution imaging/ fluorescent probe/ mitochondria/ living cell imaging
本文制备的新型探针用于标记活细胞, 所使用的细胞均为HeLa细胞. 染色方法为: 将状态较好的HeLa细胞在培养箱(37 ℃, 5%CO2)中接种在30 mm的共聚焦皿中, 探针1与探针2分别与活细胞在培养箱中共孵育15 min, 孵育好后在室温下用缓冲剂PBS冲洗3遍, 继而使用商用共聚焦显微镜(A1 R MP+, Nikon, Japan)记录图像, 结果如图3所示. 相对于探针1的共聚焦图像, 加了HSA的混合液探针2的共聚焦图像的荧光信号更强, 且定位更准确, 背景更加干净. 图 3 用探针1和探针2标记的HeLa细胞的共聚焦显微荧光图像, 比例尺为10 μm (a)探针1的荧光成像; (b)探针2的荧光成像 Figure3. Confocal images of HeLa cells labeled with probe 1 and probe 2. Scale bar is 10 μm: (a) Confocal image of HeLa cells labeled with probe 1; (b) confocal image of HeLa cells labeled with probe2.
从图3可以看出, 探针的两种形式都可以对线粒体进行标记, 但是相比较而言, 探针2的定位效果更好, 图像的信噪比更高, 这与图2(c)的光谱结果一致, 因此选用探针2对线粒体进行成像. 在此基础上, 对标记的条件进行了多项优化, 例如孵育时间和探针浓度等. 经过多次反复测试发现, 当孵育时间为15 min时, 已有充足的探针分子进入细胞, 且具有良好的染色效果. 此外多次浓度测试结果表明, 在1 ml培养基中, 使用1 μl染料(染料原始浓度为0.2 mM)荧光图像效果最好, 即探针的最佳浓度为0.2 μM. 为了研究探针2的定位效果, 使用商用的线粒体探针Mito Tracker Green FM(M7514, Thermo Fisher; 激发波长490 nm, 发射波长516 nm)和探针2进行共定位实验. 把探针2和Mito Tracker Green FM同时放置在已接种好HeLa细胞的共聚焦皿内, 置于细胞培养箱孵育15 min后, 在室温条件下用PBS进行洗涤, 可以减少背景干扰. 然后在商用的共聚焦显微镜下进行观察, 分别用488 nm的光激发Mito Tracker Green FM和561 nm的光激发探针2, 结果如图4所示. 从图4(a)可以看到, 虽然商用的探针Mito Tracker Green FM能够有效地标记线粒体, 但是线粒体周围还有较强的背景信号, 图像的信噪比较差. 图4(b)为探针2标记线粒体的图像, 从图像上可以看到探针2不仅能有效地标记上线粒体而且线粒体周围几乎没有背景信号, 这说明图像的信噪比要好于商用染料. 为了进一步研究探针2的定位效果, 将探针2与商用探针进行共定位对比, 图4(c)所示为两种染料共定位重叠后的图(绿色和红色叠加后呈黄色), 黄色区域越多说明探针2的定位效果越接近商用染料, 此处用皮尔逊相关系数$ {R}_{r} $来表示探针和示踪剂的重合度, $ {R}_{r} $值越大荧光共定位效果越好, 计算得到皮尔逊相关系数$ {R}_{r}=\text{0.769} $, 该数值表明探针2在线粒体的定位效果良好, 几乎与示踪剂Mito Tracker Green重合. 而且在图4(c)中还可以看到图片的背景略显绿色, 这主要是商用染料背景信号较强造成的, 进一步说明探针2不仅有接近商用染料的线粒体靶向性, 而且具有优于商用染料的信噪比. 图 4 用Mito Tracker Green FM和探针2共处理的HeLa细胞的共定位图像, 比例尺为10 μm (a) Mito Tracker Green FM标记的细胞图像; (b)探针2标记的细胞图像; (c)图(a)和图(b)两者的重合 Figure4. Co-localization images of Hela cells treated with Mito Tracker Green FM and Probe 2. Scale bar is 10 μm: (a) Image of Mito Tracker Green FM; (b) image of probe 2; (c) overlay of image (a) and (b).
23.2.STED成像 -->
3.2.STED成像
首先需要确定探针2的损耗光(STED光)波长(本文使用的STED系统中, STED光可选择的波长有592, 660和775 nm). 根据STED的原理, STED光的波长应尽量避开激发谱[36], 避免STED光激发荧光. 参考图2的结果, 探针2的激发峰在561 nm处, 发射峰在683 nm附近. 592 nm与激发峰太近, 因此, STED光可选的波长为660和775 nm. 分别用1 μW 561 nm的激发光, 20 mW 660 nm的STED光和20 mW 775 nm的STED光激发样品, 结果如图5所示, 从图5(a)可以看到, 当用561 nm波长的激光激发样品时, 荧光信号较强; 图5(b)是用660 nm的光照射样品得到的结果, 荧光信号也比较强, 用其作为STED光会对样品二次激发, 严重影响超分辨成像的质量, 因此660 nm的光不宜作为探针2的损耗光; 由图5(c)可以看出, 用775 nm的光照射样品, 几乎没有发射荧光, 也就是说, 775 nm的光不会造成二次激发, 可以作为探针2的损耗光. 图 5 三种不同波长的光单独照射样品时的细胞图像 (a) 561 nm的光照射; (b) 660 nm的光照射; (c) 775 nm的光照射 Figure5. Cell images illuminated by light of different wavelengths: (a) Illuminated by light of 561 nm; (b) illuminated by light of 660 nm; (c) illuminated by light of 775 nm.
接下来, 对探针2进行STED成像研究. 本实验所有的STED超分辨实验均在商用的STED显微镜(Leica SP8, Leica, Germany)上完成, 该系统使用了80 MHz且脉冲频率可调的超连续谱激光作为激发光. 根据探针的激发和发射谱(如图2所示), 以及图5的结果, 探针2的激发波长为561 nm, 选用波长为775 nm的脉冲光作为STED光. 实验中选用100 × /NA 1.4的STED专用物镜(HCX PL APO CS2 100× 1.40 oil, Leica Microsystems)进行成像, 探测器的光谱接收范围为650—750 nm. 将HeLa细胞与探针2孵育后进行STED成像, 用功率为1 μW的561 nm激光激发样品, 用不同功率的775 nm的STED光作为损耗光, 成像结果如图6所示. 结果表明, 当损耗光功率为19.75 mW时, 图像的分辨率可以达到90 nm(如图6(b)和图6(e)所示). 与共聚焦图像(图6(a)和图6(d)所示)相比, 原本不能区分的线粒体内膜可以区分开来, 并且可以看到线粒体内部脊的细微结构. 进一步将损耗光功率增加到39.5 mW, 可以更清楚地观察到线粒体内部脊的结构(图6(c)和图6(f)所示), 最高分辨率可达62 nm. 图 6 用探针2标记HeLa的共聚焦和STED图像, 比例尺为500 nm (a)共聚焦图像; (b)损耗光功率为19.75 mW时线粒体的STED图像; (c)损耗光功率为39.5 mW时的线粒体STED图像; (d)?(f)分别为图(a)?(c)中划线部分对应的信号曲线和分辨率 Figure6. Confocal and STED images of HeLa cells labeled with probe 2. Scale bar is 500 nm: (a) Confocal image; (b) STED image of mitochondria obtained with 19.75 mW STED light; (c) STED image of mitochondria obtained with 39.5 mW STED light; (d)?(f) normalized signal intensity profiles along the lines in (a)?(c) respectively as well as the spatial resolutions.
在进一步的实验中, 评估了探针在STED成像中的性能. 测量了探针2在不同功率下的发射强度, 研究了发射损耗效率, 如图7(a)所示, 探针的发射强度随着损耗光强度的增强而迅速降低. 当功率为10 mW时, 探针的受激辐射损耗比接近85%, 而后荧光强度减弱速度明显变得缓慢; 当功率为30 mW时, 达到95%的损耗效率, 高效的发射损耗比对提高探针的STED纳米成像的横向分辨率非常有利, 至此功率的进一步增加并没有进一步提高损耗比. 为了研究探针2在775 nm STED光下的饱和受激辐射功率, 测量了不同功率下775 nm STED光获得的横向分辨率, 结果如图7(b)所示. 根据(2)式可以拟合计算得出, 探针2在775 nm STED光下的饱和功率为$ {P}_{\rm{sat}}=3.5\;{\rm{mW}} $(功率密度为1.1 MW·cm–2, 损耗光束的环形光斑区域面积3.18 × 10–9 cm2). 图 7 STED光功率对成像能力的影响 (a)探针2的受激辐射损耗效率; (b)增加损耗功率情况下获得的STED图像的分辨率 Figure7. Effect of STED power on imaging performance: (a) Stimulated emission depletion efficiency of Probe 2; (b) resolution of STED images obtained at increased depletion power.
23.3.抗光漂白测试 -->
3.3.抗光漂白测试
STED成像对荧光探针的选择比共聚焦等其他成像技术有更高的要求, 抗光漂白特性是最重要的参考因素之一. 使用功率为19.75 mW的STED光对染色处理好的样品进行多次扫描, 测试探针的抗光漂白特性, 结果如图8所示. 图8中的内插图给出了在第一次扫描后的荧光图像, 此时的荧光信号较强. 经过180次扫描后, 依然可以清楚地看到完整的细胞形态. 经过长达360次扫描后, 虽然细胞结构开始变得模糊, 但仍然可以看到细胞的荧光信号, 其信号强度相对第一次和180次扫描后有明显减弱. 结果表明, 所制备的新型探针具有良好的抗光漂白特性, 适用于长时间STED成像. 图 8 探针的抗光漂白测试结果. 内插图分别为对单个细胞第1次扫描、第180次扫描和第360次扫描后得到的图像, 比例尺为10 μm Figure8. Results of bleaching test. Inset pictures are the images of single cell obtained after the first scan, 180 th scan, and 360 th scan. Scale bar is 10 μm.