Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging

We demonstrate stimulated emission depletion (STED) microscopy implemented in a laser scanning confocal microscope using excitation light derived from supercontinuum generation in a microstructured optical fiber. Images with resolution improvement beyond the far-field diffraction limit in both the lateral and axial directions were acquired by scanning overlapped excitation and depletion beams in two dimensions using the flying spot scanner of a commercially available laser scanning confocal microscope. The spatial properties of the depletion beam were controlled holographically using a programmable spatial light modulator, which can rapidly change between different STED imaging modes and also compensate for aberrations in the optical path. STED fluorescence lifetime imaging microscopy is demonstrated through the use of time-correlated single photon counting.     

Axial coding in full-field microscopy using three-dimensional structured illumination implemented with no moving parts

We report a simple optical setup to produce both axial and lateral structured illumination through a single objective lens. With a minimum of six full-field images obtained without moving either the sample or the microscope objective, 100 nm diameter fluorescent beads can be localized axially with an accuracy of 50 nm in a 1.76-microm-thick layer. We show that this axial localization improvement can easily be combined with classical lateral structured illumination, so that lateral resolution enhancement by a factor of 2 is maintained.     

Scanning probe microscope with interchangeable AFM-FFM and STM heads

Summary A scanning probe microscope operating in air with interchangeable atomic force-friction force (AFM-FFM) and electronic-tunnelling (STM) heads is presented. Our AFM operates in the so-called contact mode and utilizes the optical-lever detection method which allows simultaneous measurement of the topography as well as the lateral force. The set-up also contains an optical microscope to control both the sample and the probe laser spot on the cantilever. The experimental method to change from AFM to STM operation is based on the use of the probe laser beam and the optical microscope. The maximum scanning area is (24×24) μm² and it is well embraced in the optical-microscope visual field. The microscope attains atomic resolution in air in both AFM and STM configuration. Its performance is demonstrated on the surface of different samples. In honour of Prof. Fausto Fumi on the occasion of his retirement from teaching.     

超高分辨光学相干层析成像技术与材料检测应用

本文报道了一种超高分辨率谱域光学相干层析成像(SD-OCT)系统. 该系统基于超连续谱激光光源并截取部分光谱作为宽带光源, 其中心波长为665 nm, 光谱半高全宽(FWHM) 230 nm. 系统轴向分辨率0.9 μm, 轴向扫描速率28600行/秒, 横向分辨率3.9 μm, 横向视场1 mm, 最大成像深度0.6 mm(空气中). 利用研制的超高分辨率SD-OCT系统, 对不同型号的工业砂纸精细结构进行了成像, 并与普通SD-OCT的成像结果进行对比, 充分展示了研制系统在材料无损检测中优势.     

光学显微镜与CCD监视器组合的原子力显微镜

研制了一种与光学显微镜结合并配置 CCD 监视器的原子力显微镜,可同时获得样品的原子力显微镜图象及光学图象.已能分辨出5纳米的精细结构,最大扫描范围可达2μm.文中给出了本仪器获得的一些样品图象结果.     

Two-photon fluorescence isotropic-single-objective microscopy

Two-photon excitation provides efficient optical sectioning in three-dimensional fluorescence microscopy, independently of a confocal detection. In two-photon laser-scanning microscopy, the image resolution is governed by the volume of the excitation light spot, which is obtained by focusing the incident laser beam through the objective lens of the microscope. The light spot being strongly elongated along the optical axis, the axial resolution is much lower than the transverse one. In this Letter we show that it is possible to strongly reduce the axial size of the excitation spot by shaping the incident beam and using a mirror in place of a standard glass slide to support the sample. Provided that the contribution of sidelobes can be removed through deconvolution procedures, this approach should allow us to achieve similar axial and lateral resolution.     

High-resolution optical coherence tomography over a large depth range with an axicon lens

In optical coherence tomography, axial and lateral resolutions are determined by the source coherence length and the numerical aperture of the sampling lens, respectively. Whereas axial resolution can be improved by use of a broadband light source, there is a trade-off between lateral resolution and focusing depth when conventional optical elements are used. We report on the incorporation of an axicon lens into the sample arm of an interferometer to overcome this limitation. Using an axicon lens with a top angle of 160 degrees , we maintained 10-microm or better lateral resolution over a focusing depth of at least 6 mm. In addition to having high lateral resolution, the focusing spot has an intensity that is approximately constant over a greater depth range than when a conventional lens is used.     

A shaped annular beam tri-heterodyne confocal microscope with good anti-environmental interference capability

A shaped annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a confocal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the confocal microscope into three sub-paths (signals). Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confocal microscope and its lateral resolution can be better than 0.2 $\mu $m for $\lambda =632.8$~nm, the numerical aperture of the lens of the microscope is NA $=0.85$, and the normalized radius $\varepsilon =0.5$.     

适用于表面分析的小型低能电子显微镜

介绍并表征了一套单法兰集成的低能电子显微镜. 这套显微镜中采用了10o偏转角的磁分束器,从而有利于将其集成到一个10 in的法兰上. 电子光学系统中的一些修正单元被简化,以使其结构简单,容易操作. 样品被置于地电位,方便各静电透镜浮在高电压上. 通过几个实验展示了这台显微镜在典型的低能电子成像、低能电子衍射和光电子成像模式下的性能. 低能电子成像的空间分辨率为51 nm. 利用飞秒激光做光源,相应的非线性光电子发射过程使得这台设备非常适合进行光学近场现象的观察,并获得110 nm的光电子成像空间分辨率.     

Super‐resolution deep imaging with hollow Bessel beam STED microscopy

Stimulated emission depletion (STED) microscopy has become a powerful imaging and localized excitation method, breaking the diffraction barrier for improved spatial resolution in cellular imaging, lithography, etc. Because of specimen‐induced aberrations and scattering distortion, it is a great challenge for STED to maintain consistent lateral resolution deep inside specimens. Here we report on deep imaging STED microscopy using a Gaussian beam for excitation and a hollow Bessel beam for depletion (GB‐STED). The proposed scheme shows an improved imaging depth of up to about 155 μm in a solid agarose sample, 115 μm in polydimethylsiloxane, and 100 μm in a phantom of gray matter in brain tissue with consistent super resolution, while standard STED microscopy shows a significantly reduced lateral resolution at the same imaging depth. The results indicate the excellent imaging penetration capability of GB‐STED, paving the way for deep tissue super‐resolution imaging and three‐dimensional precise laser fabrication.

     

Demonstration of a no-moving-parts axial scanning confocal microscope using liquid crystal optics

For the first time, to the best of authors’ knowledge, a no-moving-parts axial scanning confocal microscope (ASCM) system is designed and demonstrated using a combination of a large diameter liquid crystal (LC) lens and a classical microscope objective lens. By electrically controlling the 5 mm diameter LC lens, the 633 nm wavelength focal spot is moved continuously over a 48 μm range with a measured 3-dB axial resolution of 3.1 μm using a 0.65 numerical aperture (NA) micro-objective lens. The ASCM is successfully used to image an Indium Phosphide (InP) twin square optical waveguide sample with a 10.2 μm waveguide pitch and 2.3 μm height and width. Using fine analog electrical control of the LC lens, a super-fine sub-wavelength axial resolution of 270 nm is demonstrated. The proposed ASCM can be useful in various precision three-dimensional (3D) imaging and profiling applications.     

线照明并行谱域光学相干层析成像系统与缺陷检测应用研究

针对玻璃缺陷在线无损检测的迫切需求,本文报道了一种基于线照明并行谱域光学相干层析成像系统的大视场检测系统.该系统采用快速面阵CMOS相机,单次拍摄即可获取完整的横截面(B-scan)图像.基于线照明面阵探测器的并行谱域光学相干层析成像系统,可以同时获取沿线照明方向各位置处的深度分辨信息,避免了横向扫描机构的应用.研制系统的轴向分辨率为17.9μm,并行方向上的横向分辨率55.7μm,扫描方向上的横向分辨率为24.8μm,轴向扫描速率为128 000 A-scan/s,横向视场为32 mm,空气中成像深度大于6 mm,成像灵敏度达到62 dB以上.利用研制的线照明并行谱域光学相干层析成像系统,开展了不同类型玻璃表面及其内部缺陷的检测应用研究.     

Nanometer-scale probing of optical and thermal near-fields with an apertureless NSOM

We have used a home-made apertureless near-field scanning optical microscope (ANSOM) for mapping nanometric steps between SiC and gold regions under visible (λ=655 nm) and infrared (λ=10.6 μm) illumination. The images, obtained with a signal demodulation at the tip oscillation frequency and at higher harmonics, clearly show optical contrasts with a subwavelength resolution of about 30 nm. Other images recorded in the visible on a YBa₂Cu₃O₇ crystal indicate that the tip used in our experiments is able to reveal polarization effects. We also present a near-field thermal optical microscope (NTOM) which operates without any external illumination. In this new kind of microscope, the laser source which is usually used to excite the evanescent waves, is replaced by a simple heating of the sample. The electromagnetic radiation locally scattered by the tip comes from the thermal radiation. Our results with this new technique prove a 200 nm lateral resolution.     

Theoretical assessment of optical resolution enhancement and background fluorescence reduction by three-dimensional nonlinear structured illumination microscopy using stimulated emission depletion

Three-dimensional structured illumination microscopy (SIM) enlarges frequency cutoff laterally and axially by a factor of two, compared with conventional microscopy. However, its optical resolution is still fundamentally limited. It is necessary to introduce nonlinearity to enlarge frequency cutoff further. We propose three-dimensional nonlinear structured illumination microscopy based on stimulated emission depletion (STED) effect, which has a structured excitation pattern and a structured STED pattern, and both three-dimensional illumination patterns have the same lateral pitch and orientation. Theoretical analysis showed that nonlinearity induced by STED effect, which causes harmonics and contributes to enlarging frequency cutoff, depends on the phase difference between two structured illuminations and that the phase difference of π is the most efficient to increase nonlinearity. We also found that undesirable background fluorescence, which degenerates the contrast of structured pattern and limits the ability of SIM, can be reduced by our method. These results revealed that optical resolution improvement and background fluorescence reduction would be compatible. The feasibility study showed that our method will be realized with commercially available laser, having 3.5 times larger frequency cutoff compared with conventional microscopy.     

SPDM: light microscopy with single-molecule resolution at?the?nanoscale

Far-field fluorescence techniques based on the precise determination of object positions have the potential to circumvent the optical resolution limit of direct imaging given by diffraction theory. In order to use localization to obtain structural information far below the diffraction limit, the ‘point-like’ components of the structure have to be detected independently, even if their distance is lower than the conventional optical resolution limit. This goal can be achieved by exploiting various photo-physical properties of the fluorescence labeling (‘spectral signatures’). In first experiments, spectral precision distance microscopy/spectral position determination microscopy (SPDM) was limited to a relatively small number of components to be resolved within the observation volume. Recently, the introduction of photoconvertable molecules has dramatically increased the number of components which can be independently localized. Here, we present an extension of the SPDM concept, exploiting the novel spectral signature offered by reversible photobleaching of fluorescent proteins. In combination with spatially modulated illumination (SMI) microscopy, at the present stage, we have achieved an estimated effective optical resolution of approximately 20 nm in the lateral and 50 nm in the axial direction, or about 1/25th–1/10th of the exciting wavelength.     

Three‐dimensional optical laser lithography beyond the diffraction limit

Direct laser writing has become a versatile and routine tool for the mask‐free fabrication of polymer structures with lateral linewidths down to less than 100 nm. In contrast to its planar counterpart, electron‐beam lithography, direct laser writing also allows for the making of three‐dimensional structures. However, its spatial resolution has been restricted by diffraction. Clearly, linewidths and resolutions on the scale of few tens of nanometers and below are highly desirable for various applications in nanotechnology. In visible‐light far‐field fluorescence microscopy, the concept of stimulated emission depletion (STED) introduced in 1994 has led to spectacular record resolutions down to 5.6 nm in 2009. This review addresses approaches aiming at translating this success in optical microscopy to optical lithography. After explaining basic principles and limitations, possible depletion mechanisms and recent lithography experiments by various groups are summarized. Today, Abbe's diffraction barrier as well as the generalized two‐photon Sparrow criterion have been broken in far‐field optical lithography. For further future progress in resolution, the development of novel tailored photoresists in combination with attractive laser sources is of utmost importance.     

Multiple imaging axis microscopy improves resolution for thick-sample applications

The multiple imaging axis microscope (MIAM) is a wide-field optical microscope that observes a sample simultaneously from multiple directions without requiring the sample to be rotated or tilted. The prototype is capable of high-resolution imaging of the interior of a 300-microm-diameter sample consisting of fluorescent microbeads suspended in an agarose gel. Compared with a single-axis system, the MIAM can achieve a reduction of the axial point-spread function elongation by a factor of 5.8 and a 3.5-fold improvement in volume resolution by simple linear image combination techniques.     

Field-enhanced scanning optical microscope

We present experimental results of an imaging technique that uses as a local probe the optical field enhanced at the junction of a scanning tunneling microscope illuminated by a p-polarized laser beam. Images of highly oriented pyrolithic graphite, recorded at a constant height mode, show a lateral optical resolution of as much as 10 nm. Approach curves exhibit sensitivity on a subnanometer scale of the optical signal to the tip-sample distance, yielding the ultrahigh vertical resolution reached in the images.     

High-Reflectivity A1GaN/A1N Distributed Bragg Reflector in Ultraviolet Region

Thirty-pair Alo.3 Gao.T N/A1N distributed Bragg reflectors centred at 32Ohm are designed and grown on sapphire substrates by metalorganic chemical vapour deposition. No cracks are observed in the main area of the 2-inch wafer except for about 4 mm margin under an optical microscope. Regular stack of alternating layers is shown by scanning electron microscopy. Clear two-dimensional growth steps and very low surface roughness are shown by atomic force microscopy （AFM）. Well-defined periodicity is shown by high resolution x-ray diffraction. High refiectivity of 93% at 313nm with a bandwidth of 13nm is obtained.     

Three dimensional accurate morphology measurements of polystyrene standard particles on silicon substrate by electron tomography

Polystyrene latex (PSL) nanoparticle (NP) sample is one of the most widely used standard materials. It is used for calibration of particle counters and particle size measurement tools. It has been reported that the measured NP sizes by various methods, such as Differential Mobility Analysis, dynamic light scattering (DLS), optical microscopy (OM), scanning electron microscopy (SEM) and atomic force microscopy (AFM), differ from each other. Deformation of PSL NPs on mica substrate has been reported in AFM measurements: the lateral width of PSL NPs is smaller than their vertical height. To provide a reliable calibration standard, the deformation must be measured by a method that can reliably visualize the entire three dimensional (3D) shape of the PSL NPs. Here we present a method for detailed measurement of PSL NP 3D shape by means of electron tomography in a transmission electron microscope. The observed shape of the PSL NPs with 100 nm and 50 nm diameter were not spherical, but squished in direction perpendicular to the support substrate by about 7.4% and 12.1%, respectively. The high difference in surface energy of the PSL NPs and that of substrate together with their low Young modulus appear to explain the squishing of the NPs without presence of water film.