Improving signal levels in intravital multiphoton microscopy using an objective correction collar

Multiphoton microscopy has enabled biologists to collect high-resolution images hundreds of microns into biological tissues, including tissues of living animals. While the depth of imaging exceeds that possible from any other form of light microscopy, multiphoton microscopy is nonetheless generally limited to depths of less than a millimeter. Many of the advantages of multiphoton microscopy for deep tissue imaging accrue from the unique nature of multiphoton fluorescence excitation. However, the quadratic relationship between illumination level and fluorescence excitation makes multiphoton microscopy especially susceptible to factors that degrade the illumination focus. Here we examine the effect of spherical aberration on multiphoton microscopy in fixed kidney tissues and in the kidneys of living animals. We find that spherical aberration, as evaluated from axial asymmetry in the point-spread function, can be corrected by adjustment of the correction collar of a water immersion objective lens. Introducing a compensatory positive spherical aberration into the imaging system decreases the depth-dependence of signal levels in images collected from living animals, increasing signal by up to 50%.     

In vivo fluorescence microscopy of neuronal activity in three dimensions by use of voltage-sensitive dyes

We report in vivo imaging of neuronal electrical activity from superficial layers of the mouse barrel cortex. The measurements have approximately 16-microm spatial and 3-ms temporal resolution and reach depths of 150 microm below the cortical surface. The depth-dependent differential-fluorescence optical sections of activity are consistent with known cortical architecture and represent an important step toward in vivo measurement of functioning complex neural networks. Our observations employ a custom gradient-index lens probe and voltage-sensitive dye fluorescence; the use of epi-illumination rather than dark-field illumination provides the dramatic signal-to-noise improvement necessary for fast three-dimensional imaging.     

Development of confocal X-ray fluorescence (XRF) microscopy at the Cornell high energy synchrotron source

A confocal X-ray fluorescence microscope was built at the Cornell High Energy Synchrotron Source (CHESS) to obtain compositional depth profiles of historic paintings. The microscope consists of a single-bounce, borosilicate monocapillary optic to focus the incident beam onto the painting and a commercial borosilicate polycapillary lens to collect the fluorescent X-rays. The resolution of the microscope was measured by scanning a variety of thin metal films through this confocal volume while monitoring the fluorescence signal. The capabilities of the technique were then probed using test paint microstructures with up to four distinct layers, each having a thickness in the range of 10–80 microns. Results from confocal XRF were compared with those from stand-alone XRF and visible light microscopy of the paint cross-sections. A large area, high-resolution scanner is currently being built to perform 3D scans on moderately sized paintings. PACS 29.30.Kv; 68.37.Yz; 41.50.+h     

基于电动可调焦透镜的大范围快速光片显微成像

搭建了一种基于电动可调焦透镜(electrically tunable lens)的大范围快速光片荧光显微成像系统.通过引入电动可调焦透镜与一维振镜以实现成像物平面和光片位置的快速移动,再结合高速s CMOS完成快速光片荧光显微成像.另外实验中通过改善光路与提升动态成像质量,实现了大范围扫描并减少了伪像.最终对成像性能进行测试,本系统的纵向分辨率和横向分辨率分别达到约5.5μm和约0.7μm,单幅图像稳定成像的速度约为275 frames/s,成像深度可超过138μm,能满足对具有一定尺寸的生物样本进行实时清晰成像的需求.     

Pupil-segmentation-based adaptive optical correction of a high-numerical-aperture gradient refractive index lens for two-photon fluorescence endoscopy

The intrinsic aberrations of high-NA gradient refractive index (GRIN) lenses limit their image quality as well as field of view. Here we used a pupil-segmentation-based adaptive optical approach to correct the inherent aberrations in a two-photon fluorescence endoscope utilizing a 0.8 NA GRIN lens. By correcting the field-dependent aberrations, we recovered diffraction-limited performance across a large imaging field. The consequent improvements in imaging signal and resolution allowed us to detect fine structures that were otherwise invisible inside mouse brain slices.     

Multi-color miniature dual-axis confocal microscope for point-of-care pathology

We present a miniature microelectromechanical systems-based dual-axis confocal microscope capable of spatially coregistered fluorescence and reflectance imaging at multiple wavelengths. This device has a 10 mm diameter scan head with a 2 mm diameter tip for convenient use during surgery to guide tumor resection. The microscope has an adjustable focal depth of 20-200 micrometers and is capable of imaging with an axial resolution of 9 micrometers and in-plane resolution of 4 micrometers over a field of view of 450×450 micrometers. Simultaneous two-color imaging of individual optical sections is achieved by using a pair of grating-prism assemblies to compensate for chromatic dispersion in the 2 mm diameter gradient index relay lens at the distal tip of the device. Experimental measurements of the axial response of the microscope, as well as two-color images of a reflective bar target and fresh mouse brain tissues, demonstrate the performance of our device and its potential for multicolor in vivo optical sectioning microscopy.     

方形自聚焦透镜5×5阵列的成像特性

采用离子交换工艺和精密加工技术制作了方形自聚焦透镜5×5阵列,并对其成像特性进行研究。结果表明：方形自聚焦透镜阵列相对于圆形孔径阵列而言,能有效增大填充系数,提高受光面积,其多重像和综合像的像质均匀,成像质量好。分析了导致多重像偏离和综合像变形的原因,并给出解决这一问题的关键技术。     

梯度折射率透镜在口腔内窥镜中的设计与应用

作为口腔内窥镜的摄像镜头，利用梯度折射率透镜质量轻、体积小、结构简单、分辨率高、成像景深大的优点，研制出一套适合医患交流及进行口腔检查与治疗的医疗装置。采用修正折射率剖面的梯度折射率透镜, 降低系统渐晕，减小畸变,获取口腔内部图像，经CCD光电转换装置转换成电信号，再将电信号经过数字信号处理以后，将该数字信息存储于计算机内部或在显示器上显示出来。口腔内窥镜是集梯度折射率透镜、光学原理、光学材料与加工、微电子、数字成像技术等为一体的新型医疗设备。特别是采用经修正的梯度折射率透镜制造的口腔内窥镜成像清晰, 在图像畸变、景深等方面优于现有产品，而且便于对患者的各种信息进行管理、查询、输出等。     

自聚焦透镜的三维相干成像特性分析

运用傅里叶光学方法，分析并推导了自聚焦透镜的三维相干成像公式。研究表明，虽然自聚焦透镜中介质折射率是径向渐变折射率分布，不同于薄透镜中的均匀分布，但是由于它的三维相干成像性质却类似于薄透镜，从而使自聚焦透镜可以在成像和光学信息处理系统中有效地代替薄透镜，实现光学系统的微型化。     

非圆柱形发散梯度折射率棒透镜光线传播轨迹的解析解族

本文研究光线在具有非圆柱形等折射率面的发散型梯度折射率棒透镜中的传播规律,提出在轴向弱非匀条件(即dg~(-1)(z)/dz<<1下近轴子午光线轨迹的一种解析表达式.从该解析式的解析解,棒透镜梯度参数g(z)所满足的条件出发,导出棒透镜的折射率分布族.文中给出了两个线性无关的光线传播轨迹的解析解族,并以一种发散型棒透镜为例讨论了近轴成像特性.     

球面端面的锥形梯度折射率透镜的近轴成像特性

本文根据光线在球面上的折射公式及光线微分方程,研究了光线经两端面为球面的锥形梯度折射率透镜的传播和变换,基于光线传递ABCD矩阵,提出了球面端面的锥形梯度折射率透镜的一种等效光学系统.文中给出了该透镜的主平面、焦平面和焦距计算公式,以及近轴成像高斯公式.当锥度为零时即得到球面端面的柱形或径向梯度折射率透镜的相应结果.     

Simple equivalent systems for GRIN lenses in inhomogeneous medium

Simple single-lens equivalent systems for graded-index （GRIN） lenses in inhomogeneous medium obtained using matrix optics are proposed in this letter. Due to its simplicity, the equivalent optical system enables quick analysis of the imaging properties of GRIN lens rod immersed in inhomogeneous medium. This facilitates the optical analysis of complicated optoelectronics systems in inhomogeneous medium utilizing GRIN lens rods.     

基于旋转扫描探头的OCT内窥成像系统设计

为了同时获取样品的表面和深度信息,研究光学相干层析的成像原理,建立了基于光学相干层析技术的内窥系统,实现了旋转扫描成像,系统的工作波长为1 310 nm,工作带宽为80 nm.理论推导及计算机仿真得到了系统信噪比与干涉仪的分光比、反射率之间的关系并分析了理论分辨率和探测深度.提出外径为5 mm的内窥镜扫描探头,聚焦距离为12 mm,数值孔径NA为0.47,折射率分布常量A=0.218 7.利用微型电机驱动直角棱镜实现扫描,旋转速度为25 rpm,旋转一周得到640个采样点.采用多层盖玻片和洋葱表皮作为样品进行实验分析,得到了盖玻片和洋葱的图像,横向分辨率和纵向分辨率分别为10 μm和15 μm.结果表明,设计的光学相干层析内窥系统能够用于旋转扫描成像,获取更多的组织信息.     

小鼠卵母细胞染色体三维双光子荧光图像的轴向衰减

双光子荧光显微镜作为一种高分辨光学仪器,已经被广泛应用于生物样品的非侵入式三维光学成像中。相比共聚焦显微镜,双光子荧光显微镜拥有更深的探测深度。然而,即便如此,在对较厚的生物样品进行非侵入式光学三维成像时,样品的成像质量也往往会随着探测深度的增加而下降。在临床和生物学领域对研究母性遗传起重要作用的小鼠卵母细胞拥有较大的直径(80～100 μm),吸收和散射效应较为明显。本文研究小鼠卵母细胞染色体的三维双光子荧光图像随探测深度增加图像质量的衰减程度。通过对所得图像进行轴向衰减矫正,利用体积作为参数,将矫正前后小鼠卵母细胞内染色体三维双光子荧光图像进行对比。结果表明,由于吸收和散射效应,卵母细胞存在较严重的光学轴向衰减问题,因此,对用双光子荧光三维成像手段获得的小鼠卵母细胞图像进行衰减矫正是有必要的。这为进一步精确定量的研究卵母细胞内染色体的三维构像打下良好的基础。     

基于旋转扫描探头的OCT内窥成像系统设计*

为了同时获取样品的表面和深度信息,研究光学相干层析的成像原理,建立了基于光学相干层析技术的内窥系统,实现了旋转扫描成像,系统的工作波长为1 310 nm,工作带宽为80 nm.理论推导及计算机仿真得到了系统信噪比与干涉仪的分光比、反射率之间的关系并分析了理论分辨率和探测深度.提出外径为5 mm的内窥镜扫描探头,聚焦距离为12 mm,数值孔径NA为0.47,折射率分布常量A=0.218 7.利用微型电机驱动直角棱镜实现扫描,旋转速度为25 rpm,旋转一周得到640个采样点.采用多层盖玻片和洋葱表皮作为样品进行实验分析,得到了盖玻片和洋葱的图像,横向分辨率和纵向分辨率分别为10 μm和15 μm.结果表明,设计的光学相干层析内窥系统能够用于旋转扫描成像,获取更多的组织信息.     

Superhigh numerical aperture (NA > 1.5) micro gradient-index lens based on a dual-material approach

We describe a novel scheme for obtaining a superhigh numerical aperture gradient-index (SHNA GRIN) lens from multiple thin layers of two or more materials with large refractive-index contrast. Design procedures for the lens are described, including variation of the layer thickness to achieve focusing and of the thickness limit to reduce scattering loss. We use an exact numerical solution by the finite-difference time-domain method to evaluate the lens's performance. Specific examples of a SHNA GRIN lens with a SiO2-TiO2 material system designed for fiber coupling to a nanowaveguide are shown to have focusing FWHM spot sizes of 0.53-0.7 microm at lambda =1.55 microm (corresponding to a NA of approximately 1.6-1.1) with 2.7-2.4% more loss than an ideal continuous index profile GRIN lens. With this approach, a SHNA GRIN lens with a NA of > 1.5 and a length of <20 microm can be achieved with currently available thin-film deposition techniques.     

Fabrication of a micro-optical lens using femtosecond laser 3D micromachining for two-photon imaging of bio-tissues

We report, for the first time to our knowledge, on the application of a micro-optical lens fabricated by three-dimensional (3D) femtosecond laser direct writing for two-photon fluorescence imaging of biological tissues. We show that the two-photon fluorescence images of a plant leaf tissue acquired with the micro-optical lens are comparable to that of a 5× objective lens. Our result represents an important step towards the application of micro-optical components fabricated by femtosecond laser micromachining in miniaturized nonlinear fluorescence microscopy applications, such as two-photon endoscopy.     

采用GRIN透镜的微胶囊内窥成像技术

研究了采用新型的GRIN透镜的微胶囊内窥镜成像的工作原理.根据近轴子午光线方程,用矩阵分析法分析GRIN透镜成像原理,并提出自动调焦、CCD光电转换、图像传送和无线传送电能等的实现方法.为研发微型生物医学仪器提供了必要的理论基础和重要的技术方法.     

用于运动目标探测的多通道成象系统

本文提出了一种采用自聚焦(GRIN)透镜阵列对运动目标进行探测的多通道光学成象系统.与单孔径成象系统比较,多通道成象系统具有更宽的视场和更高的运动敏感度,使之更适合于对运动目标的捕获、跟踪以及对运动量的测定.本文根据复眼的视觉机制和自聚焦透镜的成象原理,对运动目标进行了成象与测定实验.此外,初步介绍了为提高图象分辨率而采用的并行多通道处理技术.