Ultrafast photon counting applied to resonant scanning STED microscopy

To take full advantage of fast resonant scanning in super‐resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue‐to‐digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (～50 × 50 μm) with fast scanning that reduces photobleaching, and advances the time‐gated continuous wave STED technology to the usage of resonant scanning with hardware‐based time‐gating. The assembled system provides superb signal‐to‐noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant‐scanning continuous wave STED microscopy with online time‐gated detection.     

锥摆扫一体化空间相机成像模式设计

针对高分辨相机难以实现超宽覆盖的问题,本文设计了锥摆扫一体化成像模式.通过对锥摆扫一体化成像原理进行分析,建立了锥摆扫一体化成像模型;然后,设计了最佳地面轨迹模型,并分析了轨道速度、平台旋转角速度和摆扫转动角速度的关系,并基于地面轨迹给出了曝光时间、帧频的计算方案.最后,通过MATLAB进行算例仿真,并对地面覆盖宽度和...     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

Fluorescence relaxation in 3D from diffraction-limited sources of PAGFP or sinks of EGFP created by multiphoton photoconversion

The relaxation of fluorescence from diffraction‐limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo‐conversion was measured in solutions of varied viscosity (η), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point‐spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D_ap. The model incorporates two novel features that make it of broad utility. First, the model includes the no‐flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D_ap. Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D_ap and η⁻¹ were obtained for the two proteins: for PAGFP, D_aq= 89 ± 2.4 μm² s⁻¹ (mean ± 95% confidence interval), and for EGFP D_aq= 91 ± 1.8 μm² s⁻¹. In CHO cells, the application of the model yielded D_ap= 20 ± 3 μm² s⁻¹ (PAGFP) and 19 ± 2 μm² s⁻¹ (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked ‘bound fraction’ explanation of the deviation of the terminal fluorescence recovery from its pre‐bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488‐nm excitation, and the requirement for ∼1/200th the cumulative light energy to produce data of comparable signal/noise.     

共振扫描显微成像中的图像畸变校正

设计了一种校正算法用于校正双光子荧光显微镜等高速扫描成像系统中共振振镜扫描导致的图像畸变。首先对共振振镜的扫描运动建立模型,推导出非线性扫描的运动公式,进而得到图像畸变公式;然后对一块朗奇光栅样品扫描成像,设计了多峰高斯拟合算法得到光栅所有条纹的宽度变化并通过最小二乘法将条纹宽度数据拟合成一条畸变曲线;最后利用畸变曲线对图像进行校正。结果表明:采用提出的校正算法可使系统最大畸变减小到传统正弦校正方法的1/3,相对畸变减小到1/5,校正效果比传统的正弦校正法提高了2倍。由于提出的曲线拟合校正算法不用增加额外的光路,且不需要切割边缘图像,故显示了极好的图像使用效率和校正效果。     

Laser scanning photothermal microscopy: fast detection and imaging of gold nanoparticles

We report on the design and construction of a laser scanning photothermal microscope and present images of gold nanoparticles of size as small as 5 nm. Laser scanning method allows fast image acquisition at 80 μs pixel dwell time so that a 500 × 500 pixel image is acquired in 20 s. Photothermal imaging at fast time scales can have potential applications in variety of fields including tracking of biomolecular transport processes.     

Scanning moiré fringe imaging by scanning transmission electron microscopy

A type of artificial contrast found in annular dark-field imaging is generated by spatial interference between the scanning grating of the electron beam and the specimen atomic lattice. The contrast is analogous to moiré fringes observed in conventional transmission electron microscopy. We propose using this scanning interference for retrieving information about the atomic lattice structure at medium magnifications. Compared with the STEM atomic imaging at high magnifications, this approach might have several advantages including easy observation of lattice discontinuities and reduction of image degradation from carbon contamination and beam damage. Application of the technique to reveal the Burgers vector of misfit dislocations at the interface of epitaxial films is demonstrated and its potential for studying strain fields is discussed.     

3-D成像新技术——光学外差扫描全息术

介绍了一种新的3-D成像技术—光学外差扫描全息术的基本原理,以及基于光学外差扫描全息术基本原理的环形光栅光学扫描全息术,描述了其与传统的光学外差扫描全息术相比具有的优势。最后,以光学外差扫描全息术在三维空间滤波、遥感和三维混浊液体中成像应用为例,阐述了这一新技术的应用前景。     

Scanning tunneling microscope combined with scanning electron microscope

A scanning tunneling microscope (STM) has been installed in a usual scanning electron microscope (SEM) with a vacuum of 10⁻⁶ Torr. The STM image is displayed on the cathode ray tube of the SEM, 512 × 512 pixels, with a scanning rate of 80 s/picture. The spatial resolution of the STM is about 1 Å, while that of the SEM is several tens of ångströms. The combined scanning microscope covers a wide magnification range from 10 to 10⁷, where STM covers the high magnification region from 10⁵ to 10⁷.     

机载宽视场大相对孔径成像光谱仪

根据宽视场大相对孔径成像光谱仪的应用要求和技术指标,采用离轴Schwarzschild望远成像系统和双Schwarzschild光谱成像系统匹配的结构型式,设计了一个视场为28°、相对孔径为1/2.5、工作波段为0.4~1μm的机载成像光谱仪光学系统;根据双Schwarzschild光谱成像系统的像散校正条件计算了初始结构参数。然后,利用光学设计软件ZEMAX-EE进行了光线追迹和优化设计,并对设计结果进行了分析与评价。结果显示:光谱成像系统中心波长和边缘波长88%以上的能量集中在一个探测器像元内;谱线弯曲和谱带弯曲均小于像元的5%,便于光谱和辐射定标;成像光谱仪全系统在各个波长的光学传递函数均达到0.59以上,完全满足设计指标要求。该成像系统体积小、重量轻,非常适合航空遥感应用。     

Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode

We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctuation correlation spectroscopy. We compare the results obtained with the detector operating in the analog and photon counting mode. Although the dynamic range of the detector operating in the photon counting mode is superior, the performance of the analog detector is acceptable under common experimental conditions. Since most commercial laser scanning microscopes operate in the analog mode, the calculation of brightness and number of particles can be applied to data obtained with these instruments, provided that the variance is proportional to the intensity. We demonstrate that the recovered brightness of mEGFP, independent of concentration, is similar whether measured in solution or in two different cell types. Furthermore, we distinguish between mobile and immobile components, and introduce a method to correct for slow variations in intensity.     

激光共焦显微光束的偏转扫描

为了提高激光共聚焦系统的扫描速度,本文提出一种逐场扫描的场同步扫描方法。构建了激光共焦显微系统,将美国THORLABS公司的GVS002型二维检流计振镜应用于该系统,根据光学系统参数以及扫描范围要求计算振镜的整场扫描波形。借助NI公司的PCIe6353多功能数据采集卡,输出行同步的扫描波形,同时,对共焦显微系统共焦位置上针孔处的光强信号进行采集,先后扫描一幅256×256和512×512的图像,记录扫描图像和成像时间;然后,在相同的硬件结构下,以场同步的方式输出扫描波形,记录扫描图像和成像时间。实验结果表明:场同步方式扫描256×256图像的速度可提高10倍,扫描512×512图像的速度可提高5倍,且满足共焦显微成像的清晰、抗干扰能力强等要求。与行同步扫描方法相比,场同步扫描方法可以消除行与行之间转换的停留时间,在不改变硬件的情况下大幅提高扫描速度。     

Correction of image drift and distortion in a scanning electron microscopy

Continuous research on small‐scale mechanical structures and systems has attracted strong demand for ultrafine deformation and strain measurements. Conventional optical microscope cannot meet such requirements owing to its lower spatial resolution. Therefore, high‐resolution scanning electron microscope has become the preferred system for high spatial resolution imaging and measurements. However, scanning electron microscope usually is contaminated by distortion and drift aberrations which cause serious errors to precise imaging and measurements of tiny structures. This paper develops a new method to correct drift and distortion aberrations of scanning electron microscope images, and evaluates the effect of correction by comparing corrected images with scanning electron microscope image of a standard sample. The drift correction is based on the interpolation scheme, where a series of images are captured at one location of the sample and perform image correlation between the first image and the consequent images to interpolate the drift–time relationship of scanning electron microscope images. The distortion correction employs the axial symmetry model of charged particle imaging theory to two images sharing with the same location of one object under different imaging fields of view. The difference apart from rigid displacement between the mentioned two images will give distortion parameters. Three‐order precision is considered in the model and experiment shows that one pixel maximum correction is obtained for the employed high‐resolution electron microscopic system.     

Correction of distortion due to thermal drift in scanning probe microscopy

A common source of distortion in scanning probe microscope (SPM) images is “thermal drift,” the slow thermal expansion of different materials in the sample and microscope due to small changes in temperature over the course of a scan. We describe here a method for correcting this distortion by immediately following each image scan with a rescan of a small, narrow portion of the same area with the slow and fast scan axes reversed. The original, full image is corrected using a low-order polynomial mapping function, with coefficients determined by a pixel-wise comparison between the original full and rescanned partial images. We demonstrate here that this method can correctly remove distortion from a wide range of images with a precision of better than one pixel, and is also robust to common imaging artifacts. We also address some of the programming considerations that have gone into implementing this computationally intensive technique, which can now be performed using standard desktop hardware in times that range between a few seconds and a few minutes.     

Optical sectioning microscope with a binary hologram based beam scanning

We describe the development of a beam scanning microscope that can perform optical sectioning based on the principle of confocal microscopy. The scanning is performed by a laser beam diffracted from a dynamic binary hologram implemented using a liquid crystal spatial light modulator. Using the proposed scanning mechanism, unlike the conventional confocal microscopes, scanning over a two-dimensional area of the sample can be obtained without the use of a pair of galvo mirror scanners. The proposed microscope has a number of advantages, such as superior frame to frame repeatability, simpler optical arrangement, increased pixel dwell time relative to the time between two pixels, illumination of only the sample points without pulsing the laser, and absolute control over the amplitude and phase of the illumination beam on a pixel to pixel basis. The proposed microscope can be particularly useful for applications requiring very long exposure time or very large working distance objective lenses. In this paper we present experimental implementation of the setup using a nematic liquid crystal spatial light modulator and proof-of-concept experimental results.     

锥束螺旋CT半覆盖扫描重建

锥束螺旋CT能够解决长物体的检测问题,但其视场直径受限于面板探测器的宽度。为扩大锥束螺旋CT的视场直径,提出了一种螺旋CT视场区域半覆盖扫描的重建方法。扫描时,转台首先沿垂直于中心射线和转轴的方向平移一定距离,然后用普通螺旋CT的扫描方式来获得需要的投影数据。接着,利用推广的偏心锥束螺旋FDK算法进行重建,推广后的重建算法与标准的螺旋FDK算法具有同样的计算效率,而且不需要重排投影数据。实验结果表明,锥束螺旋CT半覆盖扫描能够将锥束螺旋CT的视场半径扩大1.86倍;重建图像的质量与使用大面板探测器全覆盖的标准FDK算法基本相当;由于投影数据量的减少,锥束螺旋CT半覆盖扫描的重建时间比使用大面板探测器的标准FDK算法减少了376.66s。因此,锥束螺旋CT的半覆盖扫描可以有效扩大视场直径,且具有较高的计算效率和较少的重建时间。     

扫描电化学池显微镜阿基米德螺旋快速扫描方法的研究

针对现有扫描电化学池显微镜(scanning electrochemical cell microscopy, SECCM)的扫描方法在扫描成像快速性上的不足,提出一种基于阿基米德螺旋线的新型SECCM快速扫描方法。传统跳跃扫描模式的跳跃高度是在没有先验知识的情况下靠人工经验设定的,为了避免碰撞其取值往往偏大,因此设置的扫描行程越长,消耗时间越久,速度慢、效率低。利用螺旋线轨迹预扫描快速获得待测平面的最高点,进而有效地降低了传统跳跃模式的跳跃行程,大幅提高了SECCM的扫描成像速度。此外,以螺旋线轨迹高速运动来检测最高点时,轨迹具有无冲击、因此样本运动过程无偏移,该扫描方法具有成像稳定性高的优点。通过带状金属表面成像实验表明,相对于传统跳跃扫描模式,阿基米德螺旋扫描方法在保证成像质量的同时扫描速度提高了约110%。可见提出的方法对提升SECCM扫描速度和成像质量有着重要的意义。     

基于VC++的纳米分辨远场光学共焦成像系统的扫描控制

为降低纳米分辨远场光学共焦成像系统的出错率和提高此系统的实验效率,设计了纳米分辨远场光学共焦成像系统的扫描控制子系统。纳米分辨远场光学共焦成像系统的主要组成器件为纳米移动平台,被测物体置于此平台上随平台移动而运动。如何使被测物体作有规律的运动是实验能否成功的关键。采用VC++开发平台设计的扫描自动控制子系统即为解决这一问题而开发。子系统使用方便、界面友好。     

CCD成像系统中模拟前端设计

CCD成像系统中模拟前端对系统信噪比起决定性作用。为了提高成像系统的信噪比,需要对模拟前端以及其PCB布局布线进行合理的设计。在深入分析模拟前端中各部分功能的基础上,针对目前模拟前端一般采用专用CCD视频信号处理器来实现的方案,建立了CCD视频信号处理器这个模拟数字混合器件芯片内部的电源及接地模型,并根据该模型对PCB布局布线进行了分析和设计。工程实践结果表明,在像素时钟为6 MHz时,系统的信噪比可以达到54 dB。     

基于SPI的实时时钟系统设计

介绍了一种应用于交通信号控制系统的实时时钟设计，系统硬件平台基于ARM9微处理器，使用SPI通讯功能对实时时钟进行读/写/控制。本设计硬件电路简单，可靠性高，成本低，使用方便。