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.     

Two-photon scanning microphotolysis for three-dimensional data storage and biological transport measurements

Scanning microphotolysis is a method that permits the user to select, within the scanning field of a confocal microscope, areas of arbitrary geometry for photobleaching or photoactivation. Two-photon absorption, by contrast, confers on laser scanning microscopy a true spatial selectivity by restricting excitation to very small focal volumes. In the present study the two methods were combined by complementing a laser scanning microscope with both a fast programmable optical switch and a titan sapphire laser. The efficiency and accuracy of fluorescence photobleaching induced by two-photon absorption were determined using fluorescein-containing polyacrylamide gels. At optimal conditions a single scan was sufficient to reduce the gel fluorescence by ≈40%. Under these conditions the spatial accuracy of photobleaching was 0.5±0.1 μm in the lateral ( x y ) and 3.5±0.5 μm in the axial ( z ) direction, without deconvolution accounting for the optical resolution. Deconvolution improved the accuracy values by ≈30%. The method was applied to write complex three-dimensional patterns into thick gels by successively scanning many closely spaced layers, each according to an individual image mask. Membrane transport was studied in a model tissue consisting of human erythrocyte ghosts carrying large transmembrane pores and packed into three-dimensional arrays. Upon equilibration with a fluorescent transport substrate single ghosts could be selectively photobleached and the influx of fresh transport substrate be monitored. The results suggest that two-photon scanning microphotolysis provides new possibilities for the optical analysis and manipulation of both technical and biological microsystems.     

双层衍射元件在投影式头盔光学系统设计中的应用

利用双层衍射元件设计了一款折衍混合投影式头盔光学系统。系统的衍射效率在可见光波段＞90%,提高了像面图像的对比度,增加了色彩真实性。在出瞳距离为25 mm,出瞳直径为10 mm条件下,系统的视场角为50°,有效焦距为32 mm,直径为19.2 mm,重量为7.8 g,继承了投影式头盔光学系统的轻小性特征;调制传递函数(MTF)在38 lp/mm时,边缘视场达到0.3以上,中心视场达到0.6,充分满足2.8 cm(1.1 in)彩色LCD微显示器的SXGA显示模式;系统垂轴色差为8.2 μm,场曲为0.4 m^-1,畸变为5%,成像质量满足虚拟环境和可视化训练要求。文中给出了双层衍射元件的二元面特征曲线,每周期刻蚀八个台阶时,最小特征尺寸为6.3 μm,且易于加工实现。     

单分散上转换纳米荧光微粒的荧光寿命测量

为了表征上转换纳米荧光微粒的发光特性,设计了一个可以对单个纳米微粒进行荧光寿命测量的系统。该系统首先使用基于检流计振镜的双光子显微镜系统对单分散状态的上转换纳米微粒样品进行扫描成像。然后,通过单分子荧光纳米定位算法精确找出每个纳米微粒的准确位置,再依次将激光聚焦到每个纳米微粒上,在该点施加一个500μs宽度的激光脉冲,并通过光电倍增管探测随时间变化的荧光强度信号。最后对荧光衰减曲线进行拟合,计算得到该纳米微粒的荧光寿命。实验结果表明:单个上转换纳米荧光微粒的荧光发射曲线符合单指数衰减规律,其荧光寿命为195.3μs。与之相比,聚集状态的纳米微粒的荧光寿命为358.9μs。这表明聚集状态对上转换纳米微粒的发光特性有显著影响。     

激光共振电离质谱计用光入射系统的研制

针对NINT3000质谱计的结构特点,利用高斯光学理论,推导出双透镜系统光学参数传递方程,并以此为依据设计出一套光学系统和光路调整装置,实现了激光束在离子源腔内的精确定位和聚集,焦斑半径为160μm,调节精度为10μm。目前,该系统已经安装在激光共振电离质谱计上,并实现了镥的单色双光子共振电离。     

大视场液晶自适应视网膜成像系统

设计了大视场眼底自适应成像系统,用于扩大现有视网膜自适应成像系统的视场。对人眼等晕角视场下的自适应像差校正成像进行分析,确定了波前探测与成像校正两个过程对视场的不同要求。在共光源像差探测及成像光学系统中,采用切变视场光阑的方式先后在波前探测和自适应校正成像过程中进行小-大视场切换,避免了大视场中眼波像差探测失真问题,使成像区域由200 μm扩展到500 μm。利用人眼等晕角大视场使眼底液晶自适应成像系统在不降低成像质量的前提下将成像区域扩展了2.5倍,大幅提升了该自适应成像系统在临床上应用的可行性。     

Channelling optics for high quality imaging of sensory hair

A long distance microscope (LDM) is extended by a lens and aperture array. This newly formed channelling LDM is superior in high quality, high-speed imaging of large field of views (FOV). It allows imaging the same FOV like a conventional LDM, but at improved magnification. The optical design is evaluated by calculations with the ray tracing code ZEMAX. High-speed imaging of a 2 × 2 mm(2) FOV is realized at 3.000 frames per second and 1 μm per pixel image resolution. In combination with flow sensitive hair the optics forms a wall shear stress sensor. The optics images the direct vicinity of twenty-one flow sensitive hair distributed in a quadratic array. The hair consists of identical micro-pillars that are 20 μm in diameter, 390 μm in length and made from polydimethylsiloxane (PDMS). Sensor validation is conducted in the transition region of a wall jet in air. The wall shear stress is calculated from optically measured micro-pillar tip deflections. 2D wall shear stress distributions are obtained with currently highest spatiotemporal resolution. The footprint of coherent vortical structures far away from the wall is recovered in the Fourier spectrum of wall shear stress fluctuations. High energetic patterns of 2D wall shear stress distributions are identified by proper orthogonal decomposition (POD).     

水凝胶支架的飞秒激光全息加工

为了实现以水凝胶为材料的细胞支架快速加工,将计算全息法引入传统的飞秒激光双光子加工中,并对全息图的生成方法和全息图对加工结构的影响进行了研究。首先,根据贝塞尔光波动方程和其透射函数生成贝塞尔光束全息图,分析两种参数对环形结构大小和质量的影响。然后利用生成的全息图加工得到壁厚800nm、直径为8~15μm不等的水凝胶(PEGDA)圆管结构。最后,实现了基于圆管道的水凝胶细胞支架高效快速加工,支架中圆管道壁厚800nm、直径为8μm。本文首次将飞秒激光全息加工技术应用于水凝胶三维支架加工,解决了飞秒激光单点加工效率的问题。该技术在生物医学中具有广阔的应用前景。     

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.     

The μPIVOT: an integrated particle image velocimeter and optical tweezers instrument for microenvironment investigations

A novel instrument to manipulate and characterize the mechanical environment in and around microscale objects in a fluidic environment has been developed by integrating two laser-based techniques: micron-resolution particle image velocimetry (μPIV) and optical tweezers (OT). This instrument, the μPIVOT, enables a new realm of microscale studies, yet still maintains the individual capabilities of each optical technique. This was demonstrated with individual measurements of optical trap stiffness (～70 pN μm(-1) for a 20 μm polystyrene sphere and a linear relationship between trap stiffness and laser power) and fluid velocities within 436 nm of a microchannel wall. The integrated device was validated by comparing computational flow predictions to the measured velocity profile around a trapped particle in either a uniform flow or an imposed, gravity-driven microchannel flow (R(2) = 0.988, RMS error = 13.04 μm s(-1)). Interaction between both techniques is shown to be negligible for 15 μm to 35 μm diameter trapped particles subjected to fluid velocities from 50 μm s(-1) to 500 μm s(-1) even at the highest laser power (1.45 W). The integrated techniques will provide a unique perspective toward understanding microscale phenomena including single-cell biomechanics, non-Newtonian fluid mechanics and single particle or particle-particle hydrodynamics.     

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced. The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca²⁺, or pO₂ and pCO₂. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting. The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations. Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics. Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.     

A small scanning tunnelling microscope with large scan range for biological studies

We have developed a scanning tunnelling microscope specially designed for biological applications presenting some new features: the scanner tube is mounted parallel to the surface of the sample which enables a high resolution optical microscope to be brought close to the sample when working in air or liquids. The maximum scan range is 5×20 μm with a vertical range of 20 μm and the total size of the system does not exceed 10×40 mm. The piezo-sensitivity of the scanner tube versus applied voltage was analysed by interferometry measurements and by using scanning tunnelling microscopes. We found a value for the piezoelectric constant d₁₃ of ?1·71 Å/V at low voltages (under a few volts) going up to ?2 Å/V for higher voltages. Large-scale images of a carbon grid showed a surprisingly good linearity of the scanner tube.     

PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.     

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.     

Design and implementation of a full profile sub-cm ruby laser based Thomson scattering system for MAST

A major upgrade to the ruby Thomson scattering (TS) system has been designed and implemented on the Mega-ampere spherical tokamak (MAST). MAST is equipped with two TS systems, a Nd:YAG laser system and a ruby laser system. Apart from common collection optics each system provides independent measurements of the electron temperature and density profile. This paper focuses on the recent upgrades to the ruby TS system. The upgraded ruby TS system measures 512 points across the major radius of the MAST vessel. The ruby laser can deliver one 10 J 40 ns pulse at 1 Hz or two 5 J pulses separated by 100-800 μs. The Thomson scattered light is collected at F/15 over 1.4 m. This system can resolve small (7 mm) structures at 200 points in both the electron temperature and density channels at high optical contrast; ～50% modulated transfer function. The system is fully automated for each MAST discharge and requires little adjustment. The estimated measurement error for a 7 mm radial point is <4% of T(e) and <3% of n(e) in the range of 40 eV to 2 keV, for a density of n(e)=2×10(19) m(-3). The photon statistics at lower density can be increased by binning in the radial direction as desired. A new intensified CCD camera design allows the ruby TS system to take two snapshots separated with a minimum time of 230 μs. This is exploited to measure two density and temperature profiles or to measure the plasma background light.     

利用信号拼接提高调频连续波激光测距系统的分辨力

提出了一种对等频率间隔的采样信号进行拼接来提高调频连续波激光测距系统的测距分辨力的方法。研究了调频连续波激光测距的原理,设计搭建了基于一种双干涉系统的光纤调频连续波激光测距系统。利用辅助干涉系统产生的时钟信号对测量干涉系统的信号进行等光频间隔的采样,然后对采样信号进行拼接。使用LabVIEW设计了信号错误检测处理、采样和拼接的信号处理系统。利用该测距系统进行了实验验证,结果显示,将等光频间隔的采样信号进行拼接的方法可以突破激光器扫描范围的限制,减少光源非线性的影响,从而提高系统的测距分辨力。得到结果表明,在测量距离为8.7m时,该系统的测距分辨力可达70μm,30组测量结果的重复性标准差为35μm。     

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37°C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.     

Maskless laser direct imaging lithography using a 355-nm UV light source in manufacturing of flexible fine dies

This paper describes the results of the application of laser direct imaging (LDI) lithography for dimensional tolerance improvement through surface treatment of cutting edge in the flexible fine dies (FFD). Laser direct imaging lithography was successfully performed on AISI W1-8 substrates by a specifically designed laser system using UV 355 nm light source. The characteristics of patterned line width according to conditions such as scan speed and multi pass were investigated by optical microscope. The average line width of LDIed patterns was observed to be about 1 mm. The experimental results show that the line width of patterned samples was reduced with increasing scan speed and decreasing repetition number (R/N) of scanning. This conclusion proves the feasibility in the selectively application for manufacturing flexible fine die.     

平行式Schmidt型龙虾眼X射线光学系统研究

龙虾眼X射线系统是实现大视场X射线成像的有效手段。现研制了由多组平行玻璃平板构成的Schmidt型X射线龙虾眼光学系统,开展了X射线聚焦和成像的演示实验。基于掠入射反射理论,以210μm厚的超光滑平板玻璃作为光学元件,通过堆叠的方式制作了放大倍数为1的演示系统。用8keV的X射线光源对系统进行了聚焦和实验,直径280μm的光源聚焦半高宽约为320μm,与理论模拟结果基本一致。利用X射线背光照明,得到2mm×2mm大小图样的成像。实验结果表明,该龙虾眼光学系统可以在几毫米视场下达到百微米分辨。     

CO激光在非线性晶体ZnGeP2和GaSe中的混频效应（英文）

为了获得2.15～1 500μm的相干光源,研究了CO激光在高质量非线性晶体ZnGeP2和GaSe中的混频效应。为了提高转换效率,在激光锁模方式下对CO激光器的二次谐波、和频和差频的产生进行了研究。结果显示,利用GaSe晶体和ZnGeP2晶体,调Q多谱线CO激光辐射的谱线内倍频效率分别大于0.3%和1.1%。采用ZnGeP2晶体进行倍频时,可调谐锁膜CO激光器的转换效率为12.5%。模拟结果显示,二次谐波与和频产生的输出光谱相同。相邻谱线下,和频和差频的产生过程中,基波和一次谐波可以分别在4.0～5.0μm和100～≥1 200μm(太赫兹范围)形成振荡。利用锁模CO激光器在ZnGeP2晶体中的混频效应,可以得到2.15～≥1 500μm的相干光源,同时转换效率可达到甚至高于12.5%。