超分辨率活体人眼视网膜共焦扫描成像系统

活体人眼共焦扫描成像系统的分辨率受到人眼像差、数值孔径和探测针孔尺度的限制,本文设计了一套超分辨活体人眼视网膜共焦扫描系统,采用自适应光学技术探测并校正人眼像差,结合光学超分辨技术提高系统分辨率,补偿有限尺度针孔对分辨率的影响,并获得活体人眼的实时、高分辨图像. 关键词： 超分辨 共焦扫描光学显微术 眼科光学 自适应光学     

Bimorph deformable mirror based adaptive optics scanning laser ophthalmoscope for retina imaging in vivo

A bimorph deformable mirror(DM) with a large stroke of more than 30 μm using 35 actuators is presented and characterized for an adaptive optics(AO) confocal scanning laser ophthalmoscope application. Facilitated with a Shack–Hartmann wavefront sensor, the bimorph DM-based AO operates closed-loop AO corrections for human eyes and reduces wavefront aberrations in most eyes to below 0.1 μm rms. Results from living eyes, including one exhibiting ～5D of myopia and ～2D of astigmatism along with notable high-order aberrations, reveal a practical efficient aberration correction and demonstrate a great benefit for retina imaging, including improving resolution, increasing brightness, and enhancing the contrast of images.     

Large field of view MEMS-based confocal laser scanning microscope for fluorescence imaging

Although confocal fluorescence microscopes are widely used in biology and have been proven to be promising diagnostic tools in dermatologic diagnostics, they are at present uncommon in medical practice. This is mainly due to high costs of acquisition and their large and complex outline. With the integration of a MEMS scanner we present a demonstration system of a confocal fluorescence laser scanning microscope which is affordable and portable. It has a field of view of 500 μm × 500 μm and is mainly composed of off-the-shelf components.     

共聚焦显微镜激光高速扫描控制系统设计及实现

 在传统光学扫描方法基础上,有效地将检流式与共振式光学扫描振镜结合起来,提出一种速度可达4 M/s采样率的高速激光扫描方法,并基于单片机系统设计搭建了系统控制硬件平台,编写了上位机端和下位机端应用软件。实验结果表明:该控制系统扫描速度快,性能稳定可靠,能够应用于共聚焦显微镜,实现实时扫描成像 。     

Retina imaging by using compact line scanning quasi-confocal ophthalmoscope

A compact line scanning quasi-confocal ophthalmoscope (LSO) is presented in this letter. Compared with a conventional scanning laser ophthalmoscope (SLO), the bench-top LSO significantly reduces the size, complexity, and cost of SLO utility with routine use. The LSO uses one moving part to produce high-contrast and high-resolution quasi-confocal images with nearly the same performance as a SLO. The LSO has a moderate field of view (～10 ), which enables images of the macula, the optic nerve head, and other targets to be obtained more quickly and efficiently. An image of the optic nerve head is taken in a preliminary investigation on human subjects. Individual nerve fiber bundles and vessels are resolved at a shallow depth, with a lateral resolution of nearly 10 μm.     

Subtractive imaging in confocal scanning microscopy using a CCD camera as a detector

We report a scheme for the detector system of confocal microscopes in which the pinhole and a large-area detector are substituted by a CCD camera. The numerical integration of the intensities acquired by the active pixels emulates the signal passing through the pinhole. We demonstrate the imaging capability and the optical sectioning of the system. Subtractive-imaging confocal microscopy can be implemented in a simple manner, providing superresolution and improving optical sectioning.     

Lucky averaging: quality improvement of adaptive optics scanning laser ophthalmoscope images

Adaptive optics (AO) has greatly improved retinal image resolution. However, even with AO, temporal and spatial variations in image quality still occur due to wavefront fluctuations, intraframe focus shifts, and other factors. As a result, aligning and averaging images can produce a mean image that has lower resolution or contrast than the best images within a sequence. To address this, we propose an image postprocessing scheme called "lucky averaging," analogous to lucky imaging [J. Opt. Soc. Am. 68, 1651 (1978)] based on computing the best local contrast over time. Results from eye data demonstrate improvements in image quality.     

Near-infrared laser scanning confocal microscopy and its application in bioimaging

Near-infrared (NIR) fluorescence imaging is an important imaging technology in deep-tissue biomedical imaging and related researches, due to the low absorption and scattering of NIR excitation and/or emission in biological tissues. Laser scanning confocal microscopy (LSCM) plays a significant role in the family of fluorescence microscopy. Due to the introduction of pinhole, it can provide images with optical sectioning, high signal-to-noise ratio and better spatial resolution. In this study, in order to combine the advantages of these two techniques, we set up a fluorescence microscopic imaging system, which can be named as NIR-LSCM. The system was based on a commercially available confocal microscope, utilizing a NIR laser for excitation and a NIR sensitive detector for signal collection. In addition, NIR fluorescent nanoparticles (NPs) were prepared, and utilized for fluorescence imaging of the ear and brain of living mice based on the NIR-LSCM system. The structure of blood vessels at certain depth could be visualized clearly, because of the high-resolution and large-depth imaging capability of NIR-LSCM.     

New prospects in dermatology: Fiber-based confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) constitutes an optical, noninvasive method providing visualization of tissue architecture with resolution similar to that of light microscopy. In dermatology, confocal imaging enables in vivo measurements of surface and subsurface skin microstructures. Skin annexes, as well as cutaneous cells from different epidermal layers, can be easily distinguished; their change in morphology from skin surface to the papillary dermis can be observed. Therefore, CSLM possesses a high potential for diagnostical purposes and dermatological research. The aspect of normal skin in contrast to the pathogenic state can be exposed. In our studies, we used in vivo fluorescence CSLM for morphometric analysis of healthy human skin and for imaging a number of clinically relevant inflammatory, proliferative, and neoplastic skin disorders. We report the ability to produce high-resolution histoimages of normal and pathological epidermis using this nondestructive visualization technique. Changes in keratinocyte size, shape, and morphology, as well as changes in the distribution pattern of the fluorescent emission of the dye, can be detected. Furthermore, novel fiber optic elements support a flexible handling of the rigid microscopic gadgetry. Four clinical examples of implementation were elected and instanced for demonstration.     

Combined phase-sensitive acoustic microscopy and confocal laser scanning microscopy

Combined phase-sensitive acoustic microscopy (PSAM) at 1.2 GHz and confocal laser scanning microscopy (CLSM) in reflection and fluorescence has been implemented and applied to polymer blend films and fluorescently labeled fibroblasts and neuronal cells in order to explore the prospects and the various contrast mechanisms of this powerful technique. Topographic contrast is available for appropriate samples from CLSM in reflection and, with significantly higher precision, from the acoustic phase images. Material contrast can be gained from acoustic amplitude V(z) graphs. In the case of the biological cells investigated, the optical and acoustic images are very different and exhibit different features of the samples.     

Investigation of the structure of water using a laser confocal scanning microscope

Measurements of water as the most available and vitally important element were performed using the laser confocal scanning microscope with the purpose of extending the range of its application. In this work, the measured Raman spectra of water obtained for different water samples—unprocessed, purified, and mineral, in different phase states, and after filtration and processing using different methods—are presented.     

Development of a scanning Hall probe microscope for simultaneous magnetic and topographic imaging

A scanning Hall probe microscope that is capable of observing both topographic and magnetic images simultaneously has been developed by constructing a conducting needle, used for the scanning tunneling microscope (STM) measurements, adjacent to the Hall junction of 0.6 μm square. The needle also enables the Hall probe to approach the sample without contact and to scan just above it with close proximity. Morphologies and local magnetic distributions on the surfaces of magnetic recording media, observed by our microscope, indicates that lateral spatial resolution is better than 1 μm for both STM and magnetic measurements.     

Transmission confocal laser scanning microscopy with a virtual pinhole based on nonlinear detection

We present a transmission-mode confocal laser scanning microscope system based on the use of second-harmonic generation (SHG) for signal detection. Our method exploits the quadratic intensity dependence of SHG to preferentially reveal unscattered signal light and reject out-of-focus scattered background. The SHG crystal acts as a virtual pinhole that remains self-aligned without the need for descanning.     

激光共聚焦近红外荧光扫描系统光学设计

为了实现对近红外荧光的高分辨率扫描,设计了工作在近红外光谱区的激光共聚焦光学系统。采用结构简单的凹凸双透镜物镜实现了照明光路和发射光路的设计,并采用Zemax软件进行了光学设计和仿真。实验表明:照明光路的聚焦弥散斑小于1 m,照明针孔处的聚焦光斑小于40 m,满足照明针孔的尺寸要求;发射针孔处的聚焦光斑小于10 m,满足探测针孔尺寸要求;同时照明光路和发射光路的MTF曲线的截止频率都分别满足其衍射极限分辨率的要求,照明光路在全视场空间分辨率420 lp/mm处MTF0.08,发射光路在全视场空间频率400 lp/mm处MTF0.07。     

Continuous scanning, laser imaging velocimetry

Careful exploitation of the anisotropy native to late time stratified and rotating flows permits the use of a laser scanning measurement technique to simultaneously resolve the 2D velocity field in O(100) slices. The technique relies on getting the Reynolds number from the length scale while keeping the velocity small, this provides a characteristic time scale that is sufficiently large to permit full 3D scanning through the measurement volume in a relatively short time. As the vertical velocity component of these late time stratified flows is effectively zero, all components of the deformation tensor are resolved. 3D, time resolved measurements of the vorticity and enstrophy fields associated with stratified rotating flows such as vortex dipoles, monopoles and wakes are presented.     

Fast wavelength-tunable ultra-violet laser source for confocal Fura-2AM imaging

We report a novel wavelength-flexible laser source for three-dimensional ultra-violet imaging. Based on supercontinuum generation in photonic crystal fiber, the resultant broadband laser source extended from λ = 331 nm into the visible region of the spectrum. Using an electronically-controlled filter wheel and filter set with a response time of approximately 50 ms, rapid wavelength selection was performed. The described scheme is capable of exciting the current range of ultra-violet-excited fluorophores and the simple and rapid wavelength control also provides a new approach for fast ratiometric imaging of Fura-2AM, facilitating an easy method of performing quantitative intracellular calcium concentration measurements.     

激光扫描测头视觉系统结构参数优化

线结构光视觉测量系统是激光扫描测头的重要组成部分,为了提高激光扫描测头的测量精度及可靠性,提出一种线结构光视觉系统结构参数优化设计方法。分析了影响该测量系统整体测量精度的因素以及该测量系统的结构误差模型,并建立了应用于激光扫描测头的结构约束条件,通过该约束条件建立了结构参数优化仿真系统,进而得到仿真优化后的结构参数,即最优结构参数,并设计实测实验验证其合理性。测得优化后的线结构光视觉系统测量空间点间距相对误差为0.019 8 mm,本文结果表明优化方法的有效性,并具有较好的精度。     

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.     

差动共焦显微成像高稳定三维扫描技术与系统

激光差动共焦显微镜具备高空间分辨率特点,但因其逐点扫描成像方式,扫描时间长,易受三维扫描系统不稳定和环境干扰等影响,产生系统漂移,影响仪器的空间分辨率。利用楔块机构高稳定特点,结合刹车机构的自由抱闸特性,设计了一种新型的轴向升降机构,由此构建了结构更具稳定特性的电动三维扫描系统。稳定性实验验证在搭建的激光差动共焦显微镜上进行,经过监测系统在90min内的轴向位置,轴向漂移小于50nm,与原三维扫描系统漂移140nm对比,漂移速度明显减慢,稳定性有显著提升,进而明显改善了差动共焦显微成像效果。     

Application of a femtosecond self-sustaining mode-locked Ti:sapphire laser to the field of laser scanning confocal microscopy

Developments in ultrafast Ti:sapphire laser technology can be applied in the investigation of nonlinear optical processes. We describe the application of a self-sustaining femtosecond Ti:sapphire laser as an illumination source in the field of confocal laser scanning fluorescence microscopy (LSM). We present spectra for various fluorescent stains under two-photon excitation and present LSM images of stained samples under mode-locked illumination. The potential for such a system as a non-destructive technique for studying live cells in biomedical research is discussed.