激光分光瞳共焦显微镜系统研制

为了满足生物类等样品对大工作距和高分辨率共焦显微镜的需求,将分光瞳技术与激光共焦显微技术结合应用到成像系统上。阐述了激光分光瞳共焦显微成像原理,首次成功搭建了相应的显微镜成像测量系统。理论分析和实验表明:分光瞳共焦显微技术独特的非共轴结构使系统的轴向分辨力是相同数值孔径物镜单轴系统的3倍以上,对理论高度为100nm的台阶样品进行成像测试,得到的样品三维形貌,成像质量良好。     

荧光共焦并行探测显微成像系统设计及实验

将数字微镜器件加入到荧光显微成像系统中,代替传统共焦显微系统中的照明针孔,利用其调制特性,通过在数字微镜器件上加载不同图片,实现对光束的分割.对马铃薯细胞分别进行四通道、六通道、九通道的荧光细胞探测,同时采用平面反射镜作为样品,测试得到系统的深度响应曲线,分析了系统的分辨率.实验结果表明,数字微镜器件的加入实现了从点对点共焦成像变为多点并行共焦显微成像,提高了显微成像的探测速度,同时具有较高的分辨率.     

共焦扫描荧光显微镜的信噪比与测量精度

激光扫描共焦显微术和多光子显微术等新的显微成像技术可以对厚的生物样品实现光学断层成像 ,因而在生物医学诊断领域具有重要的应用前境。在Fried的一维分辨度理论的基础上 ,系统地讨论了运用共焦扫描荧光显微术在进行光学断层成像时 ,其光学断层平面分辨度与信噪比之间的定量关系 ,建立了实际显微成像系统平面测量精度的定量计算方法。所得出的结果对于选择共焦扫描显微成像系统的最佳参数及评价所设计的显微成像系统的性能具有重要的意义。     

线结构光共焦显微成像技术的实验研究

激光共焦扫描显微镜大多采用点共焦扫描成像形式,扫描机构复杂,成本高。新型的线结构光共焦显微成像技术是对样品进行线共焦成像,只需对样品进行一维扫描就可以得到整个平面的像。设计了线结构光共焦成像实验装置,进行了线结构光共焦显微成像实验。实验结果证明了线结构光共焦成像的可行性,杂散光明显地减少,提高了成像清晰度,并且有光学层析能力。采用低照度高分辨率CCD代替价格昂贵的像增强器大大降低了系统成本。     

激光差动共焦拉曼光谱高分辨图谱成像技术进展

激光共焦拉曼光谱技术由于具有分子指纹及层析成像特性,成为探索微观分子世界的重要手段;但受原理限制,现有激光共焦拉曼光谱技术的分辨力及图谱成像能力逐渐桎梏了其发展。近年来,围绕激光共焦拉曼光谱技术性能改善方面,本研究团队基于发明的超分辨激光差动共焦技术,提出了激光差动共焦拉曼图谱成像系列新方法和新技术。系统地介绍所提激光差动共焦拉曼图谱系列测量方法及仪器化研究进展,并对未来发展方向进行了评述和展望。     

小型化望远式激光共焦扫描显微内窥镜

研制了一种激光共焦扫描显微内窥镜,采用望远式显微内窥光学系统,同时实现长距离的图像中继传输、远心f-theta光学扫描和显微内窥成像功能.二维共焦扫描由双振镜实现,低噪音扫描控制信号由嵌入式系统产生.为实现便携式应用,激光共焦扫描显微内窥镜采用小型化设计方案.首先,体内的显微内窥成像光学系统,外径尺寸为8 mm,工作长度为250.3 mm,可通过标准腹腔镜手术孔进行体内显微内窥成像;其次,采用3 mm通光孔径的小尺寸平面反射镜实现体外共焦扫描,摆动频率为100 Hz,实现快速共焦扫描;最后,激光控制和荧光探测仅通过电缆和光纤与共焦扫描显微内窥镜前端连接,减小了显微内窥镜的前端尺寸和重量.通过实验验证,本系统的成像视场为φ 600 μm,光学分辨率为2.2 μm,可采用手持式或者其他方式工作,进行体内组织的共焦扫描成像,实现微创、在体的荧光显微内窥术.     

小型化望远式激光共焦扫描显微内窥镜

研制了一种激光共焦扫描显微内窥镜,采用望近式显微内窥光学系统,同时实现长距离的图像中继传输、远心f-theta光学扫描和显微内窥成像功能.二维共焦扫描由双振镜实现,低噪音扫描控制信号由嵌入式系统产生.为实现便携式应用,激光共焦扫描显微内窥镜采用小型化设计方案.首先,体内的显微内窥成像光学系统,外径尺寸为8 mm,工作长...     

具有抗漂移特性的激光共焦拉曼光谱成像技术与系统

共焦拉曼技术结合了共焦显微技术和拉曼光谱技术,具有高分辨率、高灵敏度、可层析成像的优势,广泛应用于物理、材料科学、生物医学、文物鉴定以及刑侦等领域。由于拉曼光谱成像需要较长时间,测量中系统易受环境等因素影响产生漂移,造成离焦,而现有商用共焦拉曼光谱仪并无定焦能力,容易影响测量结果。针对此问题,研制了一种具有抗漂移能力的激光共焦拉曼光谱探测系统。在不改变共焦拉曼探测基本原理的基础上,利用拉曼轴向响应曲线最大值对应显微物镜焦面这一特性,对每个探测点进行轴向扫描,采集一定数量的轴向信号,通过曲线拟合寻找光谱强度极值位置,保证扫描过程中样品始终处于系统的焦点位置处,抑制离焦影响,改善拉曼光谱成像效果。以单层石墨烯样品进行单点测试,证明仪器在5 μm离焦范围内可以实现实时定焦,定焦后采集到的拉曼光谱强度几乎不变,具有良好的抗漂移能力;对硅台阶样品进行成像测试,结果表明成像过程中,信号强度未发生明显变化,且横向分辨率有一定改善,效果明显优于普通共焦拉曼光谱探测系统。     

基因芯片共焦扫描仪的分辨率与信噪比

介绍基因芯片共焦扫描仪的系统结构和工作原理，讨论基因芯片共焦扫描仪的光学成像系统二维分辨率与信噪比之间的定量关系。所得出的结果对于选择共焦显微成像系统的参数和评价共焦显微成像系统的性能具有重要的意义。     

基于CCD虚拟针孔探测的双轴共焦显微技术

双轴共焦显微技术具有独特的非共轴结构,与传统单轴共焦显微技术相比可利用较低数值孔径物镜实现较高的轴向分辨力,且具有工作距离大、信噪比高等优势。对基于CCD虚拟针孔(VPH)探测的双轴共焦显微成像系统的空间分辨特性进行了理论分析,并构建了相应的实验系统,对其轴向响应进行了实验验证。实验中照明物镜NAi=0.117,采集物镜NAc=0.106,θ=45°,得出系统轴向半高宽(FWHM)为2.63μm,比同等参数(NA=0.117)下单轴共焦显微系统的轴向FWHM高出约20倍。     

VC＋＋环境下激光共焦扫描显微镜的成像实现

讨论激光共焦扫描显微镜在VC＋＋环境下的图像实现过程及处理方法。     

共焦扫描光学显微镜的高分辨率

讨论了共焦扫描光学显微镜的高分辨率性质,指出共焦扫描显微镜由于采用点探测器,因而视场大大减小,信噪比大大提高,同时每幅图像逐点扫描形成,在光学系统信息能力不变的前提下,系统的空间域通带宽度增加和时域通带宽度减小。因而可成高分辨率的像,特别是其独特的深度分辨率特性使得可以实现光学断层扫描成像。给出了所研制的共焦扫描荧光显微镜所获得光学断层扫描图像     

抛光表面的亚表层损伤检测方法研究

光学元件磨削加工引入的亚表面损伤威胁着光学元件的使用性能及寿命,成为现阶段高能激光发展的瓶颈问题,特别是抛光表面光学元件的亚表面损伤检测已成为光学元件制造行业的研究热点和难点.本文结合光学共聚焦成像、层析技术、显微光学、光学散射以及微弱信号处理等技术,给出了基于光学共焦层析显微成像的光学元件亚表面损伤检测方法.分析了不同针孔大小对测量准确度的影响,并首次给出了亚表面损伤的纵向截面分布图.与腐蚀法比较结果显示:针对自行加工的同一片K9玻璃,采用本文提出的方法测得的亚表面损伤深度45 μm左右;采用化学腐蚀处理技术,对光学元件逐层刻蚀,观察得到的亚表面损伤深度50~55 μm.两者基本一致,进一步验证了本文采用的方法可以实现对光学元件亚表面损伤的定量、非破坏检测.     

A shaped annular beam tri-heterodyne confocal microscope with good anti-environmental interference capability

A shaped annular beam tri-heterodyne confocal microscope has been proposed to improve the anti-environmental interference capability and the resolution of a confocal microscope. It simultaneously detects far-, on-, and near-focus signals with given phase differences by dividing the measured light path of the confocal microscope into three sub-paths (signals). Pair-wise real-time heterodyne subtraction of the three signals is used to improve the anti-environmental interference capability, axial resolution, and linearity; and a shaped annular beam super-resolution technique is used to improve lateral resolution. Theoretical analyses and preliminary experiments indicate that an axial resolution of about 1 nm can be achieved with a shaped annular beam tri-heterodyne confocal microscope and its lateral resolution can be better than 0.2 $\mu $m for $\lambda =632.8$~nm, the numerical aperture of the lens of the microscope is NA $=0.85$, and the normalized radius $\varepsilon =0.5$.     

Dual-axes differential confocal microscopy with high axial resolution and long working distance

We discover that the slight transverse offset of a point detector results in a shift of the axial intensity response curve in a dual-axes confocal microscopy (DCM). Based on this, we propose a new dual-axes differential confocal microscopy (DDCM) with high axial resolution and long working distance, in which two point detectors are placed symmetrically about the collection axis. And a signal is obtained through the differential subtraction of two signals received simultaneously by the two point detectors. Theoretical analyses and preliminary experiments indicate that DDCM is feasible and suitable for the high precision tracing measurement of microstructures and surface contours.     

A lateral super-resolution differential confocal technology with phase-only pupil filter

In order to achieve a higher lateral resolution required for ultraprecision measurement of microstructural workpieces, phase-only pupil filtering differential confocal microscopy (PFDCM), a new approach is proposed based on the differential confocal microscopy (DCM), which uses a three-zone phase-only pupil filter with lateral super-resolution capability obtained through optimized design to change the distribution of DCM three-dimensional point spread function, so that the DCM lateral resolution is therefore significantly improved while its axial resolution is slightly improved. Preliminary experimental comparison and analyses indicate that, the lateral and axial resolutions of PFDCM are better than 0.2 μm and 2 nm, respectively, when wavelength of incidence laser beam , numerical aperture of measuring lens NA=0.85, and lateral spot size with a three-zone phase-only pupil filter G_T=0.65. It is therefore concluded that PFDCM is a new approach to further improvement of lateral resolution in laser probe measurement systems.     

Imaging biological specimens with the confocal scanning laser microscope/macroscope

A new confocal scanning laser microscope/macroscope (cslm/M) has recently been developed. It combines in one instrument the high resolution capability of a confocal scanning beam microscope for imaging small specimens, with good resolution confocal imaging of macroscopic specimens. Some of its main features include: (a) 0.25 μm lateral resolution in the microscope mode and 5 μm lateral resolution in the macroscope mode; (b) a field of view that can vary from 25 μm × 25 μm to 75,000 μm × 75,000 μm; (c) capability for acquiring large data sets from 512 × 512 pixels to 2048 × 2048 pixels; (d) 0.5 μm depth resolution in the microscope mode and 200 μm depth resolution in the macroscope mode.

In this work the cslm/M was used to image whole biological specimens (> 5 m diameter), including insects which are ideal specimens for the macroscope. Specimens require no preparation, unlike scanning electron microscope (SEM) specimens which require a conductive coating. The specimens described in this paper are too large to be imaged in their entirety by a scanning beam laser microscope, however they can be imaged by slower scanning stage microscopes. In the macroscope mode the cslm/M was used to acquire a large number (e.g. 20–40) of confocal image slices which were then used to reconstruct a three-dimensional image of the specimen. High resolution images were collected by the cslm/M by switching to the microscope mode where high numerical aperture (NA) objectives were used to image a small area of interest. Reflected-light and fluorescence images of plant and insect specimens are presented which demonstrate the morphological details obtained in various imaging modes. A process for three-dimensional visualization of the data is described and images are shown.     

反射式光纤共焦扫描成像的研究

建立了反射式光纤共焦扫描成像系统,分析了光纤－集光透镜参数Ａ及物透镜有效数值孔径等对系统成像分辨率的影响。并在此基础上选择了合适参数的透镜,获得了优化的反射式光纤共焦扫描成像系统,测试结果表明,该系统具有亚微米级横向成像能力,微米级向层析能力,成像稳定性那,它将应用于材料及生物组织三维成像检测中。     

Fiber-optic confocal microscopy using a spatial light modulator

We present a novel fiber-optic confocal microscope in which the scanning operation is achieved by use of a spatial light modulator (SLM) to sequentially illuminate individual fibers or patterns of multiple fibers. Experimental images are presented, and the optical-sectioning capability of the device is demonstrated. The novel SLM-based system is more optically efficient, achieves higher contrast, and has improved optical-sectioning capabilities compared with those of other proposed instruments for confocal microendoscopy.