Fabrication of porous copper surfaces by laser micromilling and their wetting properties

Porous copper surfaces show their great merits in the applications of chemical reaction, sound absorption and heat transfer. In this study, a laser micromilling method is proposed to fabricate porous surfaces with homogeneous micro-holes and cavities of the size about 1–15 μm on pure copper plates in a one-step process. The laser micromilling was performed by a pulsed fiber laser via the multiple–pass reciprocating scanning strategy. Based on the measurement of scanning electron microscope (SEM) and 3D laser scanning confocal microscope, the formation of surface structures was investigated together with the laser ablation mechanisms. The effects of laser processing parameters, i.e., laser fluence, scanning speed, number of scanning cycles and scanning interval, on the formation and surface morphology of porous surfaces were systematically assessed. Furthermore, the wettability of the porous copper surfaces was also evaluated by measuring the static contact angle of water. The results showed that the laser fluence played the most significant role on the formation of porous copper surfaces. The average depth and surface roughness of porous copper surfaces increased with increasing the laser fluence and number of scanning cycles while decreased with the increase in scanning interval. The scanning speed played little influence on the formation of porous copper surfaces. These results can be closely related to the variation of energy density and re-melting process during the laser micromilling process. Moreover, all the copper porous surfaces were found to be hydrophobic. The contact angle of porous copper surfaces was significantly dependent on laser fluence, but weakly affected by the scanning speed and number of scanning cycles.     

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.     

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.     

激光共聚焦显微镜的应用技术

阐述LSM510激光扫描共聚焦显微镜的工作原理及主要功能,提出LSM510激光扫描共聚焦显微镜的使用方法及荧光探针的选择。     

Fluorescence lifetime imaging by time-correlated single-photon counting

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.     

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.     

激光冲击处理对曲轴磨损性能的影响

利用激光冲击波对曲轴球墨铸铁进行表面强化处理,利用摩擦磨损试验机研究激光冲击处理球墨铸铁QT700的磨损性能,利用金相显微镜分析激光冲击处理后其表面微结构,同时采用XRD分析激光冲击处理后球墨铸铁表面马氏体与残余奥氏体的含量。结果表明,激光冲击处理后球墨铸铁具有理想的表面形貌和较高的表面硬度,其抗磨性能大幅度提高,有利于提高其疲劳寿命。     

An optical spectrometer for a confocal scanning laser microscope

An optical spectrometer for the visible range has been developed for the confocal scanning laser microscope (CSLM) Phoibos 1000. The spectrometer records information from a single point or a user-defined region within the microscope specimen. A prism disperses the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit cools the diode array, thereby reducing the detector dark current to a level, which allows integration times of up to 60 s. The spectral resolving power, λ/Δλ, ranges from 400 at λ = 375 nm to 100 at λ = 700 nm. Since the entrance aperture of the spectrometer has the same diameter as the detector aperture of the CSLM, the three-dimensional spatial resolution for spectrometer readings is equivalent to that of conventional confocal scanning, that is, down to 0.2 μm lateral and 0.8 μm axial resolution with an N.A.=1.3 objective.     

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.     

In situ laser heating in an environmental scanning electron microscope

The environmental scanning electron microscope (ESEM) offers improved capabilities for coupling a scanning electron microscope (SEM) with an in situ laser device compared with conventional SEMs. Such coupling generally enables, for example, the observation of laser glazing effects or high-temperature behaviour as well as thermal shock behaviour of materials and devices. In an ESEM, decomposition caused by high-temperature gas reactions can additionally be studied while monitoring the gaseous environment online with a mass spectrometer attached to the specimen chamber. In this work, we demonstrate the capabilities of an in situ laser system suitable for heating specimen in an in situ deformation stage, thus enabling the further study of the mechanical properties of materials far beyond temperatures accomplished by thermal heating stages.     

显微观测技术的新进展及其应用

依据显微观测技术的发展过程 ,介绍了普通的光学显微镜和 2 0世纪流行的电子显微镜 ,详细阐述了以扫描隧道显微镜和激光扫描共焦显微镜为代表的新型显微镜系列的发展 ,以及各类显微镜的基本工作原理和应用情况     

Autofluorescence of living cells

We have investigated the autofluorescence of viable mammalian cells (DU-145 and V79) with a confocal laser scanning microscope equipped with a UV laser. Our aim was to investigate the autofluorescence dependence on different treatments in mitochondria and lysosomes by using different reagents and to improve the confocal laser scanning microscope image quality by deconvolution. The following conclusions were drawn from the results: (1) not all of the autofluorescence comes from mitochondria; (2) one can significantly affect the signal which comes from the mitochondria; (3) the other organelles involved are probably lysosomes; (4) it is harder to affect the autofluorescence signal from the lysosomes than that from the mitochondria, and (5) deconvoluted autofluorescence images provide better information than undeconvoluted ones.     

Study of the focused laser spots generated by various polarized laser beam conditions

In this work, three‐dimensional near‐field imaging of the focused laser spot was studied theoretically and experimentally. In the theoretical simulation, we use the electromagnetic equivalent of the vectorial Kirchhoff diffraction integral to calculate the intensity distribution of the focal region, and a high depolarization is found in high numerical aperture systems (NA = 0.85). The experimental set‐up is based on a near‐field scanning optical microscope (NSOM) system. A high‐NA objective lens is used to focus incident light of various polarizations, and a tapered near‐field optical fibre probe of the NSOM system is used to determine the intensity of the focal field. The results show an asymmetric distribution of the focused intensity with the linear polarized laser beam.     

A confocal video-rate laser-beam scanning reflected-light microscope with no moving parts

A no-moving-parts, 30 frames/s, laser-beam scanning confocal reflected-light microscope has been developed. In principle, the technique can be extended to fluorescence and transmission light microscopy. Acousto-optic beam deflectors controlled by digital electronics move a laser beam in a 512-line interlaced 8·5 times 8·5-mm raster. The light passes through a beam splitter, enters an inverted microscope through the side camera port, and is imaged at the object by the microscope objective. Reflected light returns through the objective, exits the camera port, is reflected off the beam splitter, and is imaged on to the photocathode of an image dissector tube (IDT). Confocality is provided by raster scanning the IDT aperture coincident with the congruent image of the laser beam incident on the object. Real-time jitter-free reflected light images of a variety of biological objects have been produced. Computer-controlled alignment of the laser scan and IDT is performed in several seconds.     

Laser scanning gradient index optics microscope: submicrometre scale spectroscopy and imaging at cryogenic temperatures

A laser scanning far-field optical microscope for low-temperature imaging and spectroscopy based on gradient index optics is presented. A rod-shaped gradient index microlens is used as a zero-working-distance solid immersion objective lens. The obtained lateral resolution is 310 nm of the FWHM at a wavelength of 545 nm. A laser scanning mechanism located outside an optical cryostat enables one to achieve large scanning ranges independent of temperature. The use of the microscope for submicrometre-scale spectroscopy and low-temperature photochemistry performed on molecular J aggregates in thin polymer films is presented.     

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.     

激光扫描器及其对图像接收的影响分析

列举了激光扫描器的种类,及在激光扫描显微镜中的作用。通过对扫描振镜的分析,定量给出了反射镜反射面与振镜转轴偏心对扫描光线的影响。理论分析中定性的给出了振镜转轴的径向跳动对信号接收的影响。提出了实际使用时对扫描振镜的要求。     

Laser scanning gradient index optics microscope: submicrometre scale spectroscopy and imaging at cryogenic temperatures

A laser scanning far-field optical microscope for low-temperature imaging and spectroscopy based on gradient index optics is presented. A rod-shaped gradient index microlens is used as a zero-working-distance solid immersion objective lens. The obtained lateral resolution is 310 nm of the FWHM at a wavelength of 545 nm. A laser scanning mechanism located outside an optical cryostat enables one to achieve large scanning ranges independent of temperature. The use of the microscope for submicrometre-scale spectroscopy and low-temperature photochemistry performed on molecular J aggregates in thin polymer films is presented.     

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.     

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.