Combined Raman and continuous-wave-excited two-photon fluorescence cell imaging

We demonstrate a confocal optical microscope that combines cw two-photon-excited fluorescence microscopy with confocal Raman microscopy. With this microscope fast image acquisition with fluorescence imaging can be used to select areas of interest for subsequent chemical analysis with spontaneous Raman imaging. The distribution of the UV-absorbing fluorophore Hoechst 33342 in the apoptotic HeLa cells is measured in the combined cw two-photon-excited fluorescence and Raman microscopy modes. The 647-nm line of a Kr-ion laser is used to excite both the Raman scattering and the two-photon-excited fluorescence emission. The lateral and axial resolutions in the two imaging modes are compared by use of the Gaussian beam approximation and backprojection of the focal volume through the confocal pinhole.     

Improved axial resolution in confocal fluorescence microscopy using annular pupils

The axial image of a thick fluorescent layer is studied in a confocal microscope consisting of either circular or annular pupils. The response for circular pupils is sharper than that for annular pupils if a small pinhole detector is used. But when the size of the pinhole is larger than a certain value, the response in the latter case can become sharper than that in the former. This result implies that for a given detector of finite size, axial resolution in confocal fluorescence microscopy can be improved when an annular lens is used. Our experimental results qualitatively demonstrate the theoretical prediction. The strength of optical sectioning and the axial cross-section of the three-dimensional optical transfer function are also derived from the measured data.     

The assessment of viability in isolated rat hepatocytes subjected to cold or subzero non-freezing preservation protocols using a propidium iodide modified test

A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence--Fd) and after (total fluorescence--Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.     

Confocal microscopy with a refractive microlens-pinhole array

The stable setup of a confocal arrangement consisting of a combination of a refractive microlens and a pinhole array is presented. The focal plane of the microlenses lies at the rear surface of the substrate in the pinhole plane. By using a microscope objective one can image the stop array onto the object at a reduced size. Surface profiles of refractive and diffractive optical elements were measured with the help of this confocal microscope.     

照明物镜像差对远场衍射波前质量影响的严格矢量分析

点衍射干涉仪(PDI)中衍射参考球面波的质量受照明物镜像差和小孔的质量、状态的影响。基于矢量衍射理论,分析计算了可见光经过带像差的照明物镜聚焦后经过有限厚度具有实际电导率小孔板的衍射。分析了照明物镜像差对远场衍射波前质量的影响,确定了PDI检测极紫外光刻(EUVL)元件和系统时的最佳直径大小。分析计算得出,当用PDI检测数值孔经(NA)为0.3的系统时,采用直径大小为800nm的小孔较为适宜,其衍射波前均方根(RMS)偏差为6.51×10-5λ,强度均匀性为0.812。当用PDI检测NA为0.3的元件时,采用直径大小为500nm的小孔较为适宜,其衍射波前的RMS非对称偏差为8.40×10-5λ,强度均匀性为0.664。     

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。     

A method for calibrating the confocal volume of a confocal three-dimensional micro-x-ray fluorescence setup

The measurement of the confocal volume of a confocal three-dimensional micro-x-ray fluorescence(3 D-XRF) setup is a key step in the field of confocal 3 D-XRF analysis.With the development of x-ray facilities and optical devices,3 D-XRF analysis with a micro confocal volume will create a great potential for 2 D and 3 D microstructural analysis and accurate quantitative analysis.However,the classic measurement method of scanning metal foils of a certain thickness leads to inaccuracy.A method for calibrating the confocal volume is proposed in this paper.The new method is based on the basic content of the textbook,and the theoretical results and the feasibility are given in detail for the 3 D-XRF mono-chromatic x-ray condition and the poly-chromatic x-ray condition.We obtain a set of experimental confirmation using the polychromatic x-ray tube in the laboratory.It is proved that the sensitivity factor of the 3 D-XRF can be directly and accurately obtained in a real calibration process.     

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.     

双光子激光扫描显微镜中折射率失配引起的图象变形研究

双光子激光扫描显微镜(TPLSM)在观察生理溶液中的生物样品的结构时,水浸物镜是最好的选择.但是TPLSM在进行生物样品的三维结构观察时,需要样品和物镜在Z轴方向上做相对移动,这种移动很容易引起样品在水溶液中飘动.为了防止这种样品的漂移,往往要在水物镜和样品之间加入盖波片.由于盖波片和水及样品间的折射率失配将会导致TPLSM荧光图象的变形,给观察结果带来误差.本文主要利用TPLSM的外源探测器(ex-ternal detection)观察荧光小球的TPLSM的荧光图象,比较插入盖波片前后小球荧光图象的变化情况,研究水浸物镜在插入盖波片后折射率失配引起TPLSM图象的变形程度及共焦小孔对改进图象变形的作用.结果显示盖波片的折射率失配不仅导致TPLSM荧光图象的畸变,同时还导致TPLSM图象的荧光强度和信噪比的下降.进一步的研究表明共焦小孔可以部分改进由于折射率失配引起畸变的TPLSM荧光图象的质量.     

Chromatic confocal microscopy with a finite pinhole size

Chromatic confocal microscopy has the advantage of short measurement times because of its parallel depth scan. As most white-light sources have limited optical output power, light-efficient setups are necessary. Using an extended detection pinhole is one way to improve light efficiency. We have calculated the effect of extended pinholes in chromatic confocal setups. We found that, for certain pinhole sizes, the FWHM of the confocal signal is nearly constant over a large wavelength interval.     

Parallel confocal detection of single molecules in real time

The confocal detection principle is extended to a highly parallel optical system that continuously analyzes thousands of concurrent sample locations. This is achieved through the use of a holographic laser illumination multiplexer combined with a confocal pinhole array before a prism dispersive element used to provide spectroscopic information from each confocal volume. The system is demonstrated to detect and identify single fluorescent molecules from each of several thousand independent confocal volumes in real time.     

Effects of pinhole diameters on beam characteristics for silicon thin film optical inspection

Peak power density stability and beam-wander precision of probe laser are important factors affecting the inspection results in the precision thin film optical measurements. Pinhole is frequently used as a spatial filter in the optical inspection system. In this work, four different diameters of pinhole are investigated experimentally. It is found that pinhole diameter of 0.3 mm is considered to be a promising candidate for mounting in front of probe laser for silicon thin film optical inspection due to better peak power density stability and better beam-wander precision.     

Confocal fluorescence microscopy for minimal-invasive tumor diagnosis

The goal of the project “stereotactic laser-neurosurgery” is the development of a system for careful and minimal-invasive resection of brain tumors with ultrashort laser pulses through a thin probe. A confocal laser-scanning-microscope is integrated in the probe. In this paper, the simulation of its optical properties by a laboratory setup and the expansion by the ability for fluorescence microscopy are reported. For a valuation of the imaging properties, the point-spread-function in three dimensions and the axial depth-transfer-function were measured and thus, among other things, the resolving power and the capacity for depth discrimination were analysed. The microscope will enable intra-operative detection of tumor cells by the method of immunofluorescence. As a first model of the application in the brain, cell cultures, that fluorescein-labelled antibodies were bound to specifically, were used in this work. Due to the fluorescence signal, it was possible to detect and identify clearly the areas that had been marked in this manner, proving the suitability of the setup for minimal-invasive tumor diagnosis. Received: 11 January 1999 / Revised version: 9 August 1999 / Published online: 21 January 2000     

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     

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.     

Confocal fluorescence spectroscopy and anisotropy imaging system

We report the design and implementation of a laser scanning confocal fluorescence system with spectroscopy and anisotropy imaging capabilities. Confocal spectroscopy is achieved with a fiber pinhole that is inserted into and removed from the detection path as needed. Fluorescence anisotropy imaging is accomplished with a polarizing beam splitter placed after the conventional pinhole. Two orthogonal polarizations are detected simultaneously with balanced photomultiplier tubes. The quality of the axial sectioning that is achieved in the confocal fluorescence spectroscopy mode is demonstrated experimentally, and examples of polarization-sensitive fluorescence imaging are demonstrated in tumor cell monolayers.     

Experimental assessment of effectively probed volume in confocal XRF spectrometry using microparticles

Current approaches for assessing a confocal micro-X-rayfluorescence–probing volume involve the use of sharp knife edges, thin films, or wires, which are moved through this volume. The fluorescence radiation excited in the material of the object is measured, and profiles are built to enable the determination of the full width at half maximum in any of the three axes of the excited volume. Such approaches do not provide information on the shape of the volume, and the consequent alignment of both used lenses is made based on the position of the maxima of the registered intensity measurements. The use of particles that are smaller than the interaction volume (isolated enough to prevent the influence of nearby particles) and translated through the interaction volume (3D scan) is presented as an alternative methodology to determine the confocal probing volume. Spherical shaped uranium particles with diameter of 1–3 μm originally produced for scanning electron microscopy analysis calibration purposes were used in this study. The results obtained showed that the effectively probed confocal volume has a distinct prolate spheroidal shape that is longer in the axis of the confocal detector than it is wide on the axes of the plane perpendicular to it. The diameter in the longest axis (tilted accordingly to the angle between the two silicon drift detectors) was found to be approximately 25 μm, whereas the shorter was found about 15 μm each, with a volume of about 3,000 μm³.     

Dual-confocal fiber-optic method for absolute measurement of refractive index and thickness of optically transparent media

We present a novel noncontact optical method for absolute measurement of refractive index and thickness of optically transparent media. The method is based on a simple dual-confocal fiber-optic sensor design. It includes two independent confocal channels consisting of two identical apertureless fiber-optic-type confocal microscopes constructed by use of a single 2x2 fiber coupler. A geometrical-ray model is used to obtain the analytical dependence between the sample's refractive index and its thickness. The measurement method provides high accuracy in spatially locating the specific imaging points that correspond to the backreflected intensity peaks of the confocal responses. Thus, a simultaneous measurement of the sample refractive index and thickness is achieved.     

Interfacial shape and contact-angle measurement of transparent samples with confocal interference microscopy

A model has been developed that predicts the effective optical path through a thick, refractive specimen on a reflective substrate, as measured with a scanning confocal interference microscope equipped with a high-numerical-aperture objective. Assuming that the effective pinhole of the confocal microscope has an infinitesimal diameter, only one ray in the illumination bundle (the magic ray) contributes to the differential optical path length (OPL). A pinhole with finite diameter, however, allows rays within a small angular cone centered on the magic ray to contribute to the OPL. The model was incorporated into an iterative algorithm that allows the measured phase to be corrected for refractive errors by use of an a priori estimate of the sample profile. The algorithm was validated with a reflected-light microscope equipped with a phase-shifting laser-feedback interferometer to measure the interface shape and the 68 degrees contact angle of a silicone-oil drop on a coated silicon wafer.     

3D Particle Tracking on a Two-Photon Microscope

A 3D single-particle-tracking (SPT) system was developed based on two-photon excitation fluorescence microscopy that can track the motion of particles in three dimensions over a range of 100 μm and with a bandwidth up to 30 Hz. We have implemented two different techniques employing feedback control. The first technique scans a small volume around a particle to build up a volumetric image that is then used to determine the particle's position. The second technique scans only a single plane but utilizes optical aberrations that have been introduced into the optical system that break the axial symmetry of the point spread function and serve as an indicator of the particle's axial position. We verified the performance of the instrument by tracking particles in well-characterized models systems. We then studied the 3D viscoelastic mechanical response of 293 kidney cells using the techniques. Force was applied to the cells, by using a magnetic manipulator, onto the paramagnetic spheres attached to the cell via cellular integrin receptors. The deformation of the cytoskeleton was monitored by following the motion of nearby attached fluorescent polystyrene spheres. We showed that planar stress produces strain in all three dimensions, demonstrating that the 3D motion of the cell is required to fully model cellular mechanical responses.