Closed-loop control of a 2-D mems micromirror with sidewall electrodes for a laser scanning microscope system

This article presents the development and implementation of a robust nonlinear control scheme for a 2-D micromirror-based laser scanning microscope system. The presented control scheme, built around sliding mode control approach and augmented an adaptive algorithm, is proposed to improve the tracking accuracy in presence of cross-axis effect. The closed-loop controlled imaging system is developed through integrating a 2-D micromirror with sidewall electrodes (SW), a laser source, NI field-programmable gate array (FPGA) hardware, the optics, position sensing detector (PSD) and photo detector (PD). The experimental results demonstrated that the proposed scheme is able to achieve accurate tracking of a reference triangular signal. Compared with open-loop control, the scanning performance is significantly improved, and a better 2-D image is obtained using the micromirror with the proposed scheme.     

A new confocal scanning beam laser MACROscope using a telecentric,f-theta laser scan lens

A new confocal scanning beam system (MACROscope) that images very large-area specimens is described. The MACROscope uses a telecentric, f-theta laser scan lens as an objective lens to image specimens as large as 7·5 cm × 7·5 cm in 5 s. The lateral resolution of the MACROscope is 5 μm and the axial resolution is 200 μm. When combined with a confocal microscope, a new hybrid imaging system is produced that uses the advantages of small-area, high-speed, high-resolution microscopy (0·2 μm lateral and 0·4 μm axial resolution) with the large-area, high-speed, good-resolution imaging of the MACROscope. The advantages of the microscope/MACROscope are illustrated in applications which include reflected-light confocal images of biological specimens, DNA sequencing gels, latent fingerprints and photoluminescence imaging of porous silicon.     

Optimizing the performance of confocal point scanning laser microscopes over the full field of view

To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.     

Micromirror-based real image laser automotive head-up display

This paper reports a micromirror-based real image laser automotive head-up display (HUD), which overcomes the limitations of the previous designs by: (1) implementing an advanced display approach which is able to display sharp corners while the previous designs can only display curved lines such as to improve the display fidelity and (2) Optimizing the optical configuration to significantly reduce the HUD module size. The optical design in the HUD is simulated to choose the off-the-shelf concave lens. The vibration test is conducted to verify that the micromirror can survive 5?g. The prototype of the HUD system is fabricated and tested.     

共聚焦激光扫描荧光显微镜扫描系统研制

为适应三维光学微细加工及三维光学信息存储研究的需要,研制了共聚焦激光扫描荧光显微镜的工作台式扫描系统,扫描范围138μm×138μm.工作台采用压电陶瓷驱动器( PZT actuator)驱动的方式来获得高分辨率的位移,采用带柔性铰链的杠杆放大装置来获得较大的位移范围.描述了工作台的工作原理,并对其静态和动态性能进行了测试,实验表明这一扫描系统能很好的应用于共聚焦激光扫描荧光显微镜系统.     

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。     

Inexpensive, high-quality optical relay for use in confocal scanning beam imaging

An inexpensive, high optical-quality relay lens made up of two eyepieces arranged in an afocal assembly for use in confocal scanning laser imaging is described. In the past we have used relays, within our confocal microscopes, made up of achromats with long focal lengths (> or = 10 cm), which take up large optical tracks and suffer from significant amounts of astigmatism and curvature of field. We quantify aberrations associated with achromat and eyepiece relays using CODE V optical design and analysis software. The eyepiece relay is found to be more compact, better corrected, and not significantly more expensive than its achromat counterpart. In addition to being used to interconnect two scanning mirrors optically as well as scanning mirrors with microscope objectives, it can form part of the optics in a confocal scanning laser MACROscope-Microscope system (Biomedical Photometrics, Inc., Waterloo, Ontario, Canada). Due to design constraints, the MACROscope-Microscope system cannot incorporate a conventional wide-field microscope into its structure such as is done in most commercial confocal microscopes. The eyepiece relay is used as a stand-alone, compact optical link between the scanning mirrors and the microscope objective. This consequently makes the MACROscope-Microscope system more compact and easier to commercialize.     

Microelectromechanical diffraction gratings: Areas of applicability and prospects

The structure and specific features of operation of microelectromechanical systems, micromirror devices, and diffraction gratings controlled by an electric field are considered. Elements of microelectromechanical diffraction gratings and some applied problems solved with the use of these gratings are described. A new element of the microelectromechanical diffraction grating based on using dielectric materials with a high dielectric permeability in the gap between the electrodes is proposed. As compared with available analogs, this element has a lower control voltage, a higher clock frequency, and better processability. The characteristics of the new element and available analogs are compared.     

基于数字微镜的共焦显微系统的光路设计

详细叙述了共焦技术中的横向扫描技术,介绍了基于数字微镜(DMD)的共焦显微镜结构与原理,建立了基于DMD的并行检测系统,并且进行了光路的优化设计.实验结果表明,采用传统共焦显微镜光路时,光线出射分光棱镜时存在棱镜内表面反射问题,导致DMD上同一像素块在CCD上成两个像.在此分析了上述现象产生的原理,给出了解决此问题的方...     

Imaging modes for bilateral confocal scanning microscopy

The bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two-dimensional (2-D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow-type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non-confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time-resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2-D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real-time or can use a cooled CCD at (very) low light level for image integration over an extended period.     

Analysis of fluorescence from algae fossils of the Neoproterozoic Doushantuo formation of China by confocal laser scanning microscope

Chinese algae fossils can provide unique information about the evolution of the early life. Thin sections of Neoproterozoic algae fossils, from Guizhou, China, were studied by confocal laser scanning microscopy, and algae fossils were fluorescenced at different wavelengths when excited by laser light of 488 nm, 476 nm, and 568 nm wavelength. When illuminated by 488 nm laser light, images of the algae fossils were sharper and better defined than when illuminated by 476 nm and 568 nm laser light. The algae fossils fluoresce at a wide range of emission wavelengths. The three-dimensional images of the fluorescent algae fossils were compared with the transmission images taken by light microscope. We found that the fluorescence image of the confocal laser scanning microscope in a single optical section could pass for the transmission image taken by a light microscope. We collected images at different sample depths and made a three-dimensional reconstruction of the algae fossils. And on the basis of the reconstruction of the three-dimensional fluorescent images, we conclude that the two algae fossils in our present study are red algae.     

数字X线影像仪中激光扫描光学系统的设计

数字X线影像仪是计算机和激光技术快速发展的产物,而其中的激光扫描光学系统是其核心技术之一。针对数字X光影像仪的使用特点,对激光扫描系统中的Fθ镜头、光束扩展器、扫描器这几个关键部件进行了较详细的论述分析,提出并解决了其中一些关键问题,如光阑位置浮动对像质的影响、影响系统扫描光点大小的因素、扫描器的确定等。最后用ZEMAX光学设计软件对系统的光学性能进行了设计模拟,得到扫描光斑直径小于0.1mm、焦距和视场满足线性关系的设计结果。像质评价分析结果表明,所设计的镜头像质优良,轴上与轴外质量相当,像质达到衍射极限。     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

A high stability beam-scanning confocal optical microscope for low temperature operation

We report on the design and performance of a high stability scanning confocal microscope for optical microscopy at low temperatures. By scanning the beam in a cold objective lens system, we achieve wide fields of view without compromising image quality. Photoluminescence from single nitrogen-vacancy centers in high purity diamond is used to illustrate the imaging and stability performance of the microscope.     

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.     

改善激光共焦扫描显微镜像质的方法

为解决传统光学显微镜样本上每一点的图像都受到邻近点衍射或散射光干扰的问题,研发了一套基于C#WinForm控制平台进行连续扫描方式的激光共焦扫描显微镜(LCSM)系统,并且成功地对生物细胞进行了扫描成像。针对共焦显微镜图像像质不高的问题,提出合理选取探测器针孔直径,并通过高斯低通滤波、盲解卷积的方法,确保实现高像质。实验结果表明,基于上述方法改进后的LCSM具有较高图像质量,该方法简单易行,便于实施。     

A confocal laser microscope scanner for digital recording of optical serial sections

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illumination, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinates scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024 × 1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (～ 1 μm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.     

激光共焦扫描显微镜的光学特性研究

研究了最新发展的激光头焦扫描显微镜的下述光学特性：分辨本领及像的反差，层析分析原理及三维构像。还给出激光共焦扫描显微镜的基本光学系统及其光路按排。     

Multichannel Confocal Microscope Based on a Diffraction Focusing Multiplier with Automatic Synchronization of Scanning

Implementation of a laser scanning confocal microscope is described, where the specimen is scanned by an array of illuminating beams, which significantly increases the velocity of object image construction. The array formation is provided by using a diffractive optical element. Scanning by the array of laser beams over the specimen is performed by galvanometric scanners with moving refractive plane-parallel plates. Owing to application of such a scanning device, the beams in the illuminating channel and the signal beams in the receiving channel pass through one motionless array of confocal diaphragms; as a result, the scanning beams in the specimen plane and the signal beams in the plane of the photodetector matrix can be used without an additional synchronized pair of scanners. The proposed confocal microscope can be applied in problems that require a fast response.     

Masked illumination scheme for a galvanometer scanning high-speed confocal fluorescence microscope

High-speed beam scanning and data acquisition in a laser scanning confocal microscope system are normally implemented with a resonant galvanometer scanner and a frame grabber. However, the nonlinear scanning speed of a resonant galvanometer can generate nonuniform photobleaching in a fluorescence sample as well as image distortion near the edges of a galvanometer scanned fluorescence image. Besides, incompatibility of signal format between a frame grabber and a point detector can lead to digitization error during data acquisition. In this article, we introduce a masked illumination scheme which can effectively decrease drawbacks in fluorescence images taken by a laser scanning confocal microscope with a resonant galvanometer and a frame grabber. We have demonstrated that the difference of photobleaching between the center and the edge of a fluorescence image can be reduced from 26 to 5% in our confocal laser scanning microscope with a square illumination mask. Another advantage of our masked illumination scheme is that the zero level or the lowest input level of an analog signal in a frame grabber can be accurately set by the dark area of a mask in our masked illumination scheme. We have experimentally demonstrated the advantages of our masked illumination method in detail.