两路固体激光相干合成的实验研究

相干合成技术是获得高功率、高亮度激光输出的有效途径之一。现提出了基于角锥棱镜的两路固体激光相干合成方案;在理论分析的基础上,开展了两路固体激光器相干合成的实验研究;得到了不同占空比和出射光束直径条件下的远场光强分布。远场光强分布和条纹可见度主要受到占空比和出射光束直径的影响。随着激光光束间距的减小,相干合成后的光斑数量减少,光斑的宽度增大,相干度明显提高;出射光束直径增大时,高阶模增多,光束质量变差但合成功率增大。     

Scanning microphotolysis: A new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging

The fluorescence photobleaching method has been widely used to study molecular transport in single living cells and other microsystems while confocal microscopy has opened new avenues to high-resolution, three-dimensional imaging. A new technique, scanning microphotolysis (Scamp), combines the potential of photobleaching, beam scanning and confocal imaging. A confocal scanning laser microscope was equipped with a sufficiently powerful laser and a novel device, the ‘Scamper’. This consisted essentially of a filter changer, an acousto-optical modulator (AOM) and a computer. The computer was programmed to activate the AOM during scanning according to a freely defined image mask. As a result almost any desired pattern could be bleached (‘written’) into fluorescent samples at high definition and then imaged (‘read’) at non-bleaching conditions, employing full confocal resolution. Furthermore, molecular transport could be followed by imaging the dissipation of bleach patterns. Experiments with living cells concerning dynamic processes in cytoskeletal filaments and the lateral mobility of membrane lipids suggest a wide range of potential biological applications. Thus, Scamp offers new possibilities for the optical manipulation and analysis of both technical and biological microsystems.     

Laser surface hardening of 42CrMo cast steel for obtaining a wide and uniform hardened layer by shaped beams

For laser surface hardening (LSH) of large-sized workpieces, a wide and uniform hardened layer of a single track is pursued. In this study, two kinds of shaped laser beams were used in LSH of 42CrMo cast steel to obtain the required hardened layer. One is a stripy spot with uniform-intensity array spots and the other a stripy spot with intensity blowup in the edge of the whole array spots. As a comparison, a Gaussian laser beam was also adopted. A three-dimensional finite element model was used to simulate the thermal history of specific points by the latter shaped beam and the Gaussian laser beam. The surface morphology, microstructure, microhardness, and uniformity of hardened layers were studied. The results showed that a wider and more uniform hardened layer could be obtained using the latter shaped beam at relative higher scanning velocities and laser power. The thermal history of a material has an important effect on the microstructure and microhardness finally formed. Due to the high peak temperature and heating rate caused by the latter shaped beam, a higher value of microhardness in the transformation hardened zone was found.     

Development of a standing-wave fluorescence microscope with high nodal plane flatness

This article reports about the development and application of a standing-wave fluorescence microscope (SWFM) with high nodal plane flatness. As opposed to the uniform excitation field in conventional fluorescence microscopes an SWFM uses a standing-wave pattern of laser light. This pattern consists of alternating planar nodes and antinodes. By shifting it along the axis of the microscope a set of different fluorescent structures can be distinguished. Their axial separation may just be a fraction of a wavelength so that an SWFM allows distinction of structures which would appear axially unresolved in a conventional or confocal fluorescence microscope. An SWFM is most powerful when the axial extension of the specimen is comparable to the wavelength of light. Otherwise several planes are illuminated simultaneously and their separation is hardly feasible. The objective of this work was to develop a new SWFM instrument which allows standing-wave fluorescence microscopy with controlled high nodal plane flatness. Earlier SWFMs did not allow such a controlled flatness, which impeded image interpretation and processing. Another design goal was to build a compact, easy-to-use instrument to foster a more widespread use of this new technique. The instrument developed uses a green-emitting helium–neon laser as the light source, a piezoelectric movable beamsplitter to generate two mutually coherent laser beams of variable relative phase and two single-mode fibres to transmit these beams to the microscope. Each beam is passed on to the specimen by a planoconvex lens and an objective lens. The only reflective surface whose residual curvature could cause wavefront deformations is a dichroic beamsplitter. Nodal plane flatness is controlled via interference fringes by a procedure which is similar to the interferometric test of optical surfaces. The performance of the instrument was tested using dried and fluorescently labelled cardiac muscle cells of rats. The SWFM enabled the distinction of layers of stress fibres whose axial separation was just a fraction of a wavelength. Layers at such a small distance would lie completely within the depth-of-field of a conventional or confocal fluorescence microscope and could therefore not be distinguished by these two methods. To obtain futher information from the SWFM images it would be advantageous to use the images as input-data to image processing algorithms such as conceived by Krishnamurthi et al. (Proc. SPIE, 2655, 1996, 18–25). To minimize specimen-caused nodal plane distortion, the specimen should be embedded in a medium of closely matched refractive index. The proper match of the refractive indices could be checked via the method presented here for the measurement of nodal plane flatness. For this purpose the fluorescent layer of latex beads would simply be replaced by the specimen. A combination of the developed SWFM with a specimen embedded in a medium of matched refractive index and further image processing would exploit the full potential of standing-wave fluorescence microscopy.     

A comparison study of detecting gold nanorods in living cells with confocal reflectance microscopy and two‐photon fluorescence microscopy

Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.     

Simultaneous four-color imaging of single molecule fluorophores using dichroic mirrors and four charge-coupled devices

We developed a total-internal-reflection (TIR) fluorescence microscopy using three dichroic mirrors and four charge-coupled devices (CCDs) to detect simultaneously four colors of single-molecule (SM) fluorophores. Four spectrally distinct species of fluorophores (Alexa 488, Cy3, Cy5, or Cy5.5) were each immobilized on a different fused silica slide. A species of fluorophores on the slide was irradiated simultaneously, by two excitation beams from an Ar ion laser (488 and 514.5 nm) and a diode laser (642 nm) through TIR on the slide surface. Fluorescence emitted from the fluorophores was spectrally resolved into four components by the dichroic mirrors, and four images were generated from them simultaneously and continuously, with the four CCDs at a rate of 10 Hz. A series of images was thus obtained with each CCD. Fluorescence spots for a species were observed mainly in the series of images recorded by its respective-color CCD. In the first image in the series, we picked out the spots as continuous pixel regions that had the values greater than a threshold. Then we selected only those spots that exhibited single-step photobleaching and regarded them as SM fluorescence spots. Pixel values of SM fluorescence spots widely differed. Some SM fluorophores had pixel values smaller than the threshold, and were left unpicked. Assuming the pixel values of SM fluorescence spots differed with a Gaussian profile, we estimated the ratios of unpicked fluorophores to be less than 20% for all the species. Because of the spectral overlaps between species, we also observed cross-talk spots into CCDs other than the respective-color CCDs. These cross-talk SM fluorescence spots can be mistaken for correct species. We thus introduced the classification method and classified SM fluorescence spots into correct species in accordance with two kinds of four-dimensional signal vectors. The error rates of fluorophore classification were estimated to be less than 3.2% for all the species. Our system is suitable for the biological studies that desire to simultaneously monitor the four colors of SM fluorophores.     

High efficiency beam splitter for multifocal multiphoton microscopy

In this article we present the development of a multibeam two-photon laser scanning microscope. A new type of beam splitter to create the multitude of laser beams is described. This type of beam splitter has higher transmission and generates more uniform beams than can be achieved with the microlens approach used by other groups. No crosstalk exists between the different foci due to small temporal delays between the individual beams. The importance of dispersion compensation to obtain maximum efficiency of the microscope is discussed. With optimum compensation the fluorescence signal was raised by a factor of 14. Different modes of detecting the fluorescence signals and their effect on imaging speed and resolution are discussed.     

Total internal reflection fluorescent microscopy

This review discusses applications of fluorescence microscopy using totally internally reflected excitation light. When totally internally reflected in a transparent solid at its interface with liquid, the excitation light beam penetrates only a short distance into the liquid. This surface electromagnetic field, called the ‘evanescent wave’, can selectively excite fluorescent molecules in the liquid near the interface. Total internal reflection fluorescence (TIRF) has been used to examine the cell/substrate contact regions of primary cultured rat myotubes with acetylcholine receptors labelled by fluorescent α-bungarotoxin and human skin fibroblasts labelled with a membrane-incorporated fluorescent lipid. TIRF examination of cell/substrate contacts dramatically reduces background from cell autofluorescence and debris. TIRF has also been combined with fluorescence photobleaching recovery and correlation spectroscopy to measure the chemical kinetic binding rates and surface diffusion constant of fluorescent labelled serum protein binding (at equilibrium) to a surface.     

Double-pulse fluorescence lifetime imaging in confocal microscopy

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination.     

Picosecond-resolution fluorescence lifetime measuring system with a cw laser and a radio

A procedure for measurement of fluorescence lifetimes with picosecond time resolution is described. A cw laser beam is modulated with a standing-wave acousto-optic modulator. The modulated beam is split; one part serves as a reference beam, the other part excites the fluorescent sample. The sample flourescence and the reference beam, attenuated and delayed optically to be equal in amplitude and opposite in phase to the fluorescence, are incident onto a single photomultiplier tube. The thus achieved photodetector ac null is monitored either by an AM radio, whose intermediate-frequency signal is displayed on an oscilloscope, or by a spectrum analyzer. With 30-MHz light modulation and the radio, lifetimes could be determined with resolution better than 15 ps. With the spectrum anlyzer and 170-MHz light modulation frequency we have achieved 4-ps lifetime resolution. Correction for photomultiplier transit time versus incident wavelength is made.     

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

Data processing of the two-channel adaptive straightness measurement system based on optical activity

In this paper the adaptive noise-cancelling technique (ANC) is applied in a two-channel straightness measurement system based on optical activity. The ANC technique may get rid of the correlated noise. In this system, one laser beam serves for monitoring the environment turbulence and the other is for measurement. Because the two beams are sufficiently close to each other, the condition of correlation is satisfied. The experimental results show that the compensating effect is 30–80% after filtering. Theoretical and experimental results are given.     

分体式激光扩束系统平行度测量装置的设计

为精确测量强激光发射系统中高功率激光经分体式扩束系统后光束的传输方向,设计了一种新型分体式扩束系统输出光平行度测量装置.根据高功率激光分体式扩束系统及红外激光的特点,该装置采用高分辨率红外CCD作为监测成像设备.采用轻质高刚度的优质铝合金对装置的机械结构进行了设计,切换部件搭载在高精度线性位移平台上.基于高分辨率CCD和精密线性位移平台,该装置可较好地完成动态和静态测量.测试结果表明,该平行度测量装置工作稳定、可靠,测量精度优于2.0″;装置设计合理,实用,可为扩束系统的装调及应用提供可靠依据.     

单光源双光路激光并行共焦测量系统设计

针对传统激光并行共焦测量过程中存在的泰伯效应,提出将数字微镜器件（DMD）引入激光并行共焦测量系统来正确辨识正焦面的位置。采用了DMD作为光分束器件,从理论上验证了它是一种投影式的阵列光源,对激光分束后不会在光路方向上产生泰伯像;同时,考虑DMD不能对分束后的光线产生会聚作用,并非高效的并行光源分束器件,本文将DMD与微透镜阵列(MLA)结合构建了单光源双光路并行共焦测量系统。该系统利用DMD光路探测正焦面位置,利用微透镜阵列光路进行精确的共焦测量。实验结果表明,两种光路下的正焦面位置仅相差2 μm,在一个泰伯间距范围之内,可以较好地克服泰伯效应对激光并行共焦测量的影响,进而保证较高精度的并行共焦测量。     

Vital confocal microscopy in bone

We wished to exploit confocal microscopy for high spatial and temporal resolution vital microscopy in bone. To this end, we evolved implants with glass windows supported in titanium, which were placed in the medial proximal tibial plateau of the rabbit, and special small, self-focussing objectives (dry 10/0.25, water immersion 20/0.45, and oil immersion 45/0.65 and 120/1.0) which mated and matched to the conical window entrance section of the metal components. At intervals of up to 21 months after implant healing, these lenses were used to study live tissue using two genera of confocal microscope: multiple aperture disc, tandem scanning, microscopes for observation in reflection, and video rate confocal laser scanning microscopes for recording, mainly in the fluorescence mode. The latter allowed the study of a variety of intravenously administered substances, including fluorescein, fluorescein-dextrans, fluorescent microspheres, acridine orange, DASPMI, calcein, and tetracycline. We were able to remove blood, stain cells with fluorescent markers, and replace them into the circulation. Calcein and tetracycline bind to the mineral front in bone: this labelling was studied in progress. We observed that both substances partition and remain for long periods (at least days) in adipocytes. Further characterisation of the system used both confocal fluorescence and scanning electron microscopy methods in the study of retrieved implants. These studies showed that the subimplant cortical bone remodelled to a less compact structure with a rich microvasculature extremely close to bone. The points of attachment of bone to glass were found to involve coarse fibres, with the matrix containing large numbers of large cells: some of this tissue was cartilage and some immature bone. An amorphous, mineralised matrix was in immediate contact with glass. The results provide further confirmation of the general utility of high-scan speed confocal methodology in physiology.     

Imaging analysis of the interface between osteoblasts and microrough surfaces of laser‐sintered titanium alloy constructs

Previous work using focused ion beam (FIB) analysis of osteoblasts on smooth and microrough Ti surfaces showed that the average cell aspect ratio and distance from the surface are greater on the rough surface. In order to better interrogate the relationship between individual cells and their substrate using multiple imaging modalities, we developed a method that tracks the same cell across confocal laser scanning microscopy (CLSM) to correlate surface microroughness with cell morphology and cytoskeleton; scanning electron microscopy (SEM) to provide higher resolution for observation of nanoroughness as well as chemical mapping via energy dispersive X‐ray spectroscopy; and transmission electron microscopy (TEM) for high‐resolution imaging. FIB was used to prepare thin sections of the cell‐material interface for TEM, or for three‐dimensional electron tomography. Cells were cultured on laser‐sintered Ti‐6Al‐4V substrates with polished or etched surfaces. Direct cell to surface attachments were observed across surfaces, though bridging across macroscale surface features occurred on rough substrates. Our results show that surface roughness, cell cytoskeleton and gross morphology can be correlated with the cell‐material cross‐sectional interface at the single cell level across multiple high‐resolution imaging modalities. This work provides a platform method for further investigating mechanisms of the cell‐material interface.     

Probing rotation dynamics of biomolecules using polarization based fluorescence microscopy

Fluorescence polarization, particularly fluorescence anisotropy (FA) can be used to characterize the rotation dynamics and interactions of biomolecules. We report here fluorescence polarization microscopy based on a two-photon fluorescence microscope. Two-photon fluorescence excited by a linearly polarized fs laser beam was separated into components of parallel and perpendicular polarization and then recorded simultaneously by two detectors. From the images corresponding to different combinations of the polarization for the excitation and fluorescence photons, images of FA, or polarization difference, can be derived. It is demonstrated that FA microscopy is capable of probing rotational mobility of the fluorescent molecules and their interaction with the surroundings, but displays lower axial resolution than fluorescence intensity images. It is proved that the degraded axial resolution of FA imaging is intrinsic to the current experimental set-up. Artifacts in FA imaging of aligned molecules are also discussed.     

高精度激光光束准直系统设计

为了进一步提高激光光束用作直线基准的精度,建立了基于反射镜平动式光束稳定器以及两点式光束漂移分离法的高精度激光光束准直系统。首先,对系统中基于反射镜平动的光束稳定器进行研究,对其光束偏转原理以及影响因素进行分析,并对两点式光束漂移分离方法进行介绍。然后,对光束偏转单元的分辨力以及偏转范围、所使用的压电陶瓷非线性和迟滞特性、以及光束偏转单元的频率响应特性进行实验测试。最后,对该激光准直系统的激光光束准直精度进行测试。实验结果表明本文提出的光束偏转单元在120μrad范围内的光束偏转分辨力可以达到5 nrad,频率响应特性高于2 kHz;最终激光准直系统的准直精度在二维方向上分别达到1.9×10^-8 rad和2.1×10^-8 rad,相对于现有技术约提高了3倍,满足激光光束用作高精度直线基准的需求。     

基于二分束的三角法激光测距研究

为了实现激光测距仪小型化和低成本制作,提出了一种新型的基于高斯光束二分束的三角法激光测距方法,采用双PIN管探测器来替代传统三角法激光测距系统中的CCD或PSD光电检测单元,实现光束的二分束;利用光度学和高斯光束理论,推导出两分束的光功率比与待测距离及发射角的关系,并通过数值计算获得其关系图,进而通过测量分束比计算出目标距离。试验结果表明,本方法具有结构简单、精度高、对目标物颜色深浅不敏感等优越特性,并具有良好的应用前景。     

Reflection across plant cell boundaries in confocal laser scanning microscopy

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.