Hacking the optical diffraction limit: Review on recent developments of fluorescence nanoscopy

Subject to the diffraction limit, the resolution of conventional optical microscopy is constrained to about 200 and 500 nm in the lateral and axial planes, respectively. The advantage of optical microscopy in the life sciences over electronic microscopy, especially fluorescence microscopy, drives scientists to develop novel "hacks" to reach nanoscale resolutions by optical means. In this review, three aspects of the techniques are discussed: (1) lateral super-resolution; (2) axial super-resolution; (3) super-resolution in three dimensions. The principles of how the methods achieve the cross-barrier resolution are discussed, and recent advances in current techniques are described. With these methods, the use of fluorescence microscopy is growing quickly toward a new era: fluorescence nanoscopy that will reveal 2 orders of magnitude more information on cellular structure and dynamics.     

结构光和显微术相结合实现光学层析

针对共焦测量中单点扫描速度慢、机构复杂、光能利用率低的问题，介绍了一种利用结构光和共焦显微术相结合来实现多光束测量的新方法．通过改装显微照明光路，投射一结构光到被测物体上，获得了物体的层析像与普通显微镜成像的叠加像．该叠加像经过解码算法，实现了非零频分量的层析成像，并利用互强度理论，对该层析显微镜的层析能力和解码算法从理论上给出了表达式．数值模拟表明，该系统与针孔阵列、微透镜阵列和孔径相关相比，不仅简便可行，而且提高了速度，光能利用率也得到了明显的改善．     

基于改进SRResNet深度学习网络的低照度图像超分辨率重建方法

为解决低照度条件下低分辨率图像的超分辨率重建问题,提出一种基于改进超分辨率残差网络(super-resolution residual networks, SRResNet)深度学习网络的低照度图像超分辨率重建方法。通过将读取的图像下采样、亮度降低等处理生成低照度低分辨率图像,并将该图像与高分辨率图像作为数据对输入学习模型,以便改进SRResNet的训练数据对的生成方式,优化训练过程,从而构建面向单帧低照度彩色图像的基于改进SRResNet训练的超分辨率重建方法。实验结果表明：与现有流行的图像超分辨率重建方法相比,该方法的峰值信噪比(peak signal to noise ratio, PSNR)、结构相似性(structural similarity, SSIM)指标整体为最优,低照度环境下的超分辨率重建图像更为清晰明亮、细节更丰富,该方法训练出的深度学习网络的重建效果更好。     

超分辨率荧光显微技术——解析2014年诺贝尔化学奖

 2014 年诺贝尔化学奖授予Eric Betzig,Stefan W. Hell 和William E. Moerner3 位科学家,以表彰他们在超分辨率荧光显微成像技术方面的重大贡献。本文从显微镜分辨率的起因入手,对超分辨荧光显微技术进行了深入阐述。此外,对光学显微技术的发展前景进行展望。     

Investigation of the effects of MoO_3 buffer layer on charge carrier injection and extraction by capacitance–voltage measurement

The effects of MoO3thin buffer layer on charge carrier injection and extraction in inverted configuration ITO/ZnO/MEH-PPV(poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene))/MoO3(0,5 nm)/Ag hybrid solar cells are investigated by capacitance–voltage measurement under dark and light illumination conditions.The efficiency of charge carrier injection and extraction is enhanced by inserting 5 nm MoO3thin layer,resulting in better device performances.Charge carrier transport of the whole device is improved and the interface energy barrier is reduced by inserting 5 nm MoO3thin buffer layer.The device fill factor is increased from 54.1%to 57.5%after modifying 5 nm MoO3.Simulations and experimental results consistently show that in the forward voltage under dark,the device with the 5 nm MoO3thin layer modification generates larger value of capacitance than the device without MoO3layer.While under illumination,the device with the 5 nm MoO3layer generates smaller value of capacitance than the device without the 5 nm MoO3layer in the bias region of reverse and before the peak position of maximum capacitance(VCmax).The underlying mechanism of the MoO3anode buffer layer on device current density–voltage characteristics is discussed.     

Functionalized graphene oxide based on p-phenylenediamine as spacers and nitrogen dopants for high performance supercapacitors

p-Phenylenediamine （PPD） functionalized graphene oxide （GO） materials （PPDG） were prepared through a one-step solvothermal process and their appli-cation as supercapacitors （SCs） were studied. The PPD is not only as the spacers to prevent aggregating and re-stacking of the graphene sheets in the preparing process but also as nitrogen sources to obtain the nitrogen-doped graphene. The structures of PPDG were characterized by Fourier transformed infrared spectroscopy （FT-IR）, X-ray diffraction spectroscopy （XRD）, Raman spectroscopy and X-ray photoelectron spectroscopy （XPS） and the results show that the nitrogen-doped graphene was achieved with nitrogen content as high as 10.85 at.%. The field emission scanning electron microscopy （FE-SEM） and high resolu-tion transmission electron microscopy （HR-TEM） have confirmed that the morphologies of PPDG were looselayered with less aggregation, indicating that PPD mole-cules, as spacers, effectively prevent the graphene sheets from restacking during the solvothermal reaction. The special loose textures make PPDG materials exhibit excellent electrochemical performance for symmetric SCs with superior specific capacitance （313 F/g at 0.1 A/g）, rate capability and cycling stability. The present synthesis method is convenient and may have potential applications as ultrahigh performance SCs.     

Thermal radiation scanning tunnelling microscopy

In standard near-field scanning optical microscopy (NSOM), a subwavelength probe acts as an optical 'stethoscope' to map the near field produced at the sample surface by external illumination. This technique has been applied using visible, infrared, terahertz and gigahertz radiation to illuminate the sample, providing a resolution well beyond the diffraction limit. NSOM is well suited to study surface waves such as surface plasmons or surface-phonon polaritons. Using an aperture NSOM with visible laser illumination, a near-field interference pattern around a corral structure has been observed, whose features were similar to the scanning tunnelling microscope image of the electronic waves in a quantum corral. Here we describe an infrared NSOM that operates without any external illumination: it is a near-field analogue of a night-vision camera, making use of the thermal infrared evanescent fields emitted by the surface, and behaves as an optical scanning tunnelling microscope. We therefore term this instrument a 'thermal radiation scanning tunnelling microscope' (TRSTM). We show the first TRSTM images of thermally excited surface plasmons, and demonstrate spatial coherence effects in near-field thermal emission.     

Optical microscopy using a single-molecule light source

Rapid progress in science on nanoscopic scales has promoted increasing interest in techniques of ultrahigh-resolution optical microscopy. The diffraction limit can be surpassed by illuminating an object in the near field through a sub-wavelength aperture at the end of a sharp metallic probe. Proposed modifications of this technique involve replacing the physical aperture by a nanoscopic active light source. Advances in the spatial and spectral detection of individual fluorescent molecules, using near-field and far-field methods, suggest the possibility of using a single molecule as the illumination source. Here we present optical images taken with a single molecule as a point-like source of illumination, by combining fluorescence excitation spectroscopy with shear-force microscopy. Our single-molecule probe has potential for achieving molecular resolution in optical microscopy; it should also facilitate controlled studies of nanometre-scale phenomena (such as resonant energy transfer) with improved lateral and axial spatial resolution.     

基于SQI的超分辨率图像重建算法

提出了一种基于自商图像(Self Quotient Image-SQI)的超分辨率图像重建算法.该方法首先利用SQI提取光照不变量作为图像特征,并假设光反射分量具有分段平滑的特性,近似认为每一个小的图像块具有相同的增益系数;然后在流形学习的框架下,借助局部线性嵌套的思想构建高分辨率图像和低分辨率图像块间的关系,从而实现了超分辨率重建和图像增强.仿真结果表明,该算法有效地克服了传统方法受光照因素影响的缺点,在提高分辨率的同时克服了光照因素的影响,特别是对阴影效应的消除具有明显效果.     

近场光学与近场光学显微镜

近场光学是研究距离物体表面一个波长以内的光学现象的新型交叉学科。基于非辐射场的探测与成像原理,近场光学显微镜突破常规光学显微镜所受到的衍射极限,在超高光学分辨率下进行纳米尺度光学成像与纳米尺度光谱研究。本文将讨论近场光学的基本原理,非辐射场的探测与高分辨率的关系;光学限阈及隐失场在近场光学中的重要性;以及近场信息的获取方法。对近场光学显微镜的主要类型及相应的仪器发展,分辨率,衬度原理做一综述。同时简要介绍近场光学显微镜在超高分辨率光学成像,近场局域光谱,高密度数据存储,在生命科学,单分子光谱,量子器件发光机制等领域中的应用。     

Three-dimensional chromatin distribution in neuroblastoma nuclei shown by confocal scanning laser microscopy

The relationship between cell shape and function has long been of interest. However, although the behaviour of the cytoskeleton during the cell cycle has been studied extensively variations in the shape and three-dimensional substructure of the nucleus are less well documented. The spatial distribution of chromatin has previously been studied by a mathematical analysis of the optical densities of stained nuclei, allowing an indirect derivation of the three-dimensional distribution of chromatin. More direct information on chromatin organization can be obtained from electron-microscopic serial sections, although this is very laborious. Using an iterative deconvolution algorithm, Agard and Sedat achieved a degree of optical sectioning in conventional fluorescence microscopy and reconstructed the three-dimensional arrangement of polytene chromosomes. We report here on the three-dimensional structure of cultured mammalian cells as visualized by confocal scanning laser microscopy (CSLM). The exceptionally short depth of field of this imaging technique provides direct optical sectioning which, together with its higher resolution, makes CSLM extremely useful for studying the three-dimensional morphology of biological structures.     

碲化锌修饰掺铜硒化锌量子点的合成

以巯基丙酸（mercaptopropionic acid,MPA）作为稳定剂,在水相中合成Cu离子掺杂的ZnSe量子点（quantum dots,QDs）,并以ZnTe修饰其表面（ZnSe:Cu/ZnTe QDs）.采用X射线衍射（X-ray diffraction,XRD）、高分辨透视电子显微镜（high resolution transmission electronic microscopy,HRTEM）、紫外可见吸收光谱（ultraviolent-visible spectroscopy,UV-VIS）和光致发光(photoluminescence,PL)荧光光度计对其结构、相貌和光学特性进行表征.结果表明,合成所得荧光量子点的大小为4～6 nm;当激发波长325 nm时,荧光发射峰约为510 nm;经160 ℃热处理后,荧光发射峰会红移至540 nm左右,初步说明ZnTe的修饰会改变ZnSe:Cu量子点荧光发射峰的位置.     

Atomic force microscopy observation on nuclear reassembly in a cell-free system

Cell-free system is interesting and useful for studying nuclear assembly in mitosis. Atomic force microscopy (AFM), which is a simple way for imaging fixed reassemble nuclei with high resolution, has not been used in the cell-free system. In this paper, we put forward an air-drying sample preparation for AFM. Using AFM, we observed nuclear reassembly process within 100 nm resolution in a cell-free system. As a result, we found that the images were artifact-free, and with higher resolution compared with fluorescent optical microscope images. Furthermore, the morphology of membrane vesicles was obtained dearly, and a dynamic change of morphology during the vesides‘‘ approaching to nuclear envelope was also observed, which is enlightened to understand the mechanism of nuclear envelope assembly.     

Association of cystatin C level and cardiovascular prognosis for patients with preexisting coronary heart disease: a meta-analysis

The association of circulating levels of cystatin C (CysC) and risk of recurrent cardiovascular events in patients with preexisting coronary heart disease are uncertain. To qualitatively and quantitatively address this issue, we performed a random meta-analysis of results from prospective studies on the topic. We searched electronic and printed sources (up to 16 October 2011) using keywords and retrieved articles for seven prospective studies according to the selection criteria. Data were abstracted and meta-analysis was performed using the random-effects model (RevMan 5.0.23 software). The cohorts involved 4,576 patients with preexisting coronary heart disease (CHD) and normal or mild chronic kidney disease, and follow-up ranged from 5.6 to 40.6 months. Highest versus lowest baseline CysC level was significantly associated with increased risk of cardiovascular events (OR 2.30, 95 % CI 1.80–2.94), all-cause mortality (OR 5.69, 95 % CI 3.70–8.74), but not with recurrent MI (OR 1.75, 95 % CI 0.77–4.00). In heterogeneity testing for studies reporting cardiovascular events and all-cause mortality, no significant heterogeneity was found, and exclusion of any single study did not alter the overall finding. For risk of recurrent MI, significant heterogeneity was found among the five trials (χ ² = 13.16, P = 0.01, I ² = 70 %) and exclusion of the Taglieri et al. [12] study from the analysis resulted in a rise in relative risk (OR 2.36, 95 % CI 1.09–5.15). In conclusion, for patients with established CHD, high baseline level of CysC is associated with increased risk of cardiovascular events and all-cause death during long-term follow-up.     

Super-resolution microscopy of live cells using single molecule localization

The resolution of conventional light microscopy is insufficient for subcelluar studies. The invention of various super-resolution imaging techniques breaks the diffraction barrier and pushes the resolution limit towards the nanometer scale. Here, we focus on a category of super-resolution microscopy that relies on the stochastic activation and precise localization of single molecules. A diversity of fluorescent probes with different characteristics has been developed to achieve super-resolution imaging. In addition, with the implementation of robust localization algorithms, this family of approaches has been expanded to multi-color, three-dimensional and live cell imaging, which provides a promising prospect in biological research.     

切片技术在聚合物材料研究中的应用

将切片技术与透射电子显微镜（TEM）、原子力显微镜（AFM）、光学显微镜（LM）等快速发展的显微分析技术结合起来，可以研究聚合物材料的形貌结构、相容性和结晶，以及填料的分散及形貌结构等，有助于优化聚合物材料的制备工艺条件、配方和性能。进一步优化切片速度、温度等切片技术参数并加强与显微红外光谱等其他显微学表征手段的结合，将使切片技术在高分子材料的结构分析与性能优化中发挥更加重要的作用。     

Diffraction-unlimited all-optical imaging and writing with a photochromic GFP

Lens-based optical microscopy failed to discern fluorescent features closer than 200?nm for decades, but the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off is making nanoscale imaging routine. Reported fluorescence nanoscopy variants switch these features either with intense beams at defined positions or randomly, molecule by molecule. Here we demonstrate an optical nanoscopy that records raw data images from living cells and tissues with low levels of light. This advance has been facilitated by the generation of reversibly switchable enhanced green fluorescent protein (rsEGFP), a fluorescent protein that can be reversibly photoswitched more than a thousand times. Distributions of functional rsEGFP-fusion proteins in living bacteria and mammalian cells are imaged at <40-nanometre resolution. Dendritic spines in living brain slices are super-resolved with about a million times lower light intensities than before. The reversible switching also enables all-optical writing of features with subdiffraction size and spacings, which can be used for data storage.     

Investigation of coupled cobalt–silver nanoparticle system by plan view TEM

We present a transmission electron microscopy (TEM) investigation of a coupled cobalt and silver nanoparticle system. A plan view in situ lift-out method for preparing samples for TEM using the focused ion beam (FIB) microscope was used. This technique is used to prepare high quality TEM samples with site specificity in a short time and with a high success rate. We demonstrate the ability of the plan view sample preparation technique to provide information about an ordered system of nanoparticles which could not be observed using standard FIB cross sectioning of the sample. High resolution TEM and energy dispersive X-ray spectroscopy mapping of both cross sectional and plan view samples are presented, clearly showing the significant benefit of plan view TEM analysis for certain samples     

Photoinduced effects in amorphous semiconductor Ge(S, Se)2 chalcogenide films

The blue shift of optical transmittance edges were observed in amorphous semiconductor Ge(S, Se)₂ chalcogenide films with light illumination. The shift in well-annealed films could be recovered by annealing the films near the glass-transition temperature again. The photocrystallization was also observed in amorphous Ge(S,Se)₂ films with light illumination by the transmitting electron microscope measurement. The photoinduced phenomina of the amorphous Ge(S,Se)₂ films could be applied to designing some new kinds of optical storage materials.     

Synthesis of Li_4Ti_5O_(12) nanostructural anode materials with high charge–discharge capability

This study demonstrates a facile and efficient hydrothermal method to prepare spindle titanate(Li4Ti5O12 denoted as LTO)and/or carbon-LTO nanocomposites(CLTO),in which the LTO or C-LTO microspheres have diameters of a few micrometers,composed of numerous nanosheets with thickness of*30 nm and edge length of hundreds of nanometers.The morphology and size control of these nanoparticles could be achieved by varying experimental parameters including concentration of titanium butoxide,lithium hydroxide,and cetyltrimethylammonium bromide,as well as reaction temperature and time.These micro-nanostructures were characterized by several advanced techniques,such as transmission electron microscopy,scanning electron microscopy,X-ray diffraction,energy dispersive spectroscopic analysis,surface area,and electrochemical measurements.The LTO and C-LTO microstructures were examined in the charge–discharge capacity at a rate of 50 C,as well as the stability after 100 cycles at a rate of 10 C.The excellent capability may be attributed to good conductivity,large surface area,and stable assembly structure of such micro-nanostructures,which could be explored as a promising anode material for lithium-ion batteries.