共聚焦激光扫描荧光显微镜扫描系统研制

为适应三维光学微细加工及三维光学信息存储研究的需要,研制了共聚焦激光扫描荧光显微镜的工作台式扫描系统,扫描范围138μm×138μm.工作台采用压电陶瓷驱动器( PZT actuator)驱动的方式来获得高分辨率的位移,采用带柔性铰链的杠杆放大装置来获得较大的位移范围.描述了工作台的工作原理,并对其静态和动态性能进行了测试,实验表明这一扫描系统能很好的应用于共聚焦激光扫描荧光显微镜系统.     

Quantitative measurement of the resolution and sensitivity of confocal microscopes using line-scanning fluorescence correlation spectroscopy

Spatial resolution and the sensitivity to detect a fluorophore are the two most important optical parameters that characterize a confocal microscope. However, these are rather difficult to estimate quantitatively. We show that fluorescence correlation spectroscopy (FCS) provides an easy and reliable measure of these quantities. We modify existing schemes for performing FCS on a commercial confocal microscope to carry out these measurements, and provide an analysis routine that can yield the relevant quantities. Our method does not require any modification of the confocal microscope, yet it yields a robust measure of the resolution and sensitivity of the instrument.     

Effect of a confocal pinhole in two-photon microscopy.

We investigated the effect of a finite-sized confocal pinhole on the performance of nonlinear optical microscopes based on two-photon excited fluorescence and second-harmonic generation. These techniques were implemented using a modified inverted commercial confocal microscope coupled to a femtosecond Ti:sapphire laser. Both the transverse and axial resolutions are improved when the confocal pinhole is used, albeit at the expense of the signal level. Therefore, the routine use of a confocal pinhole of optimized size is recommended for two-photon microscopy wherever the fluorescence or harmonic signals are large.     

Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting

Multidimensional time-correlated single photon counting (TCSPC) is based on the excitation of the sample by a high-repetition rate laser and the detection of single photons of the fluorescence signal in several detection channels. Each photon is characterized by its arrival time in the laser period, its detection channel number, and several additional variables such as the coordinates of an image area, or the time from the start of the experiment. Combined with a confocal or two-photon laser scanning microscope and a pulsed laser, multidimensional TCSPC makes a fluorescence lifetime technique with multiwavelength capability, near-ideal counting efficiency, and the capability to resolve multiexponential decay functions. We show that the same technique and the same hardware can be used for precision fluorescence decay analysis and fluorescence correlation spectroscopy (FCS) in selected spots of a sample.     

Field enhancement and apertureless near-field optical spectroscopy of single molecules

We report on fluorescence enhancement in near field optical spectroscopy by apertureless microscopy. Our apertureless microscope is designed around a confocal fluorescence microscope associated with an AFM head. First, we show that the confocal microscope alone allows single molecule imaging and single molecule fluorescence analysis. When associated with the AFM head, we demonstrate, for the first time to our knowledge, that single molecule fluorescence is enhanced under the silicon tip. We tentatively attribute this effect to field enhancement under the tip.     

Field enhancement and apertureless near-field optical spectroscopy of single molecules

We report on fluorescence enhancement in near field optical spectroscopy by apertureless microscopy. Our apertureless microscope is designed around a confocal fluorescence microscope associated with an AFM head. First, we show that the confocal microscope alone allows single molecule imaging and single molecule fluorescence analysis. When associated with the AFM head, we demonstrate, for the first time to our knowledge, that single molecule fluorescence is enhanced under the silicon tip. We tentatively attribute this effect to field enhancement under the tip.     

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.     

A standard video-rate confocal laser-scanning reflection and fluorescence microscope

A confocal laser-scanning microscope (CLSM) differs from a conventional microscope by affording an extreme depth discrimination, as well as a slightly improved resolution. These features afford improved imaging, and make possible new imaging techniques. The CLSM developed at TNO has standard video-rate imaging, and is capable of working in reflection and in fluorescence mode simultaneously. Nonconfocally the laser-scanning microscope can also be used in transmission mode. In addition to the evident advantages of a fast system when searching objects or studying living objects, the time needed to produce an image of extended depth of focus and high resolution is very short. Furthermore, the high-speed averaging of many images at low laser-power levels, and the short dwelling time of the focused laser beam (60 ns) obviate quenching effects in fluorescence microscopy and prevent damage to the object. In this article the TNO-CLSM system is outlined. The most important specifications are summarized, and some representative micrographs obtained with the system are shown. Furthermore, the performance of the system is illustrated by some experimental results.     

Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy

Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico‐ to nano‐molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation spectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non‐ideal confocal volume can be experimentally determined and validated.     

改善激光共焦扫描显微镜像质的方法

为解决传统光学显微镜样本上每一点的图像都受到邻近点衍射或散射光干扰的问题,研发了一套基于C#WinForm控制平台进行连续扫描方式的激光共焦扫描显微镜(LCSM)系统,并且成功地对生物细胞进行了扫描成像。针对共焦显微镜图像像质不高的问题,提出合理选取探测器针孔直径,并通过高斯低通滤波、盲解卷积的方法,确保实现高像质。实验结果表明,基于上述方法改进后的LCSM具有较高图像质量,该方法简单易行,便于实施。     

Optic fibre bundle contact imaging probe employing a laser scanning confocal microscope

A small diameter (600 µm) fused optic fibre imaging bundle was used as a probe to compare fluorescent specimens by direct contact imaging using both a conventional fluorescence microscope and a laser scanning confocal microscope (LSCM) system. Green fluorescent polyester fibres placed on a green fluorescent cardboard background were used to model biological tissue. Axial displacement curves support the hypothesis that pinhole size in the LSCM system reduces the contribution of non‐focal plane light. Qualitative comparison showed that the LSCM system produced superior image quality and contrast over the conventional system. The results indicate that the new LSCM–probe combination is an improvement over conventional fluorescence–probe systems. This study shows the feasibility of employing such a small diameter probe in the investigation of biological function in difficult to access areas.     

Fluorescence intensity is a poor predictor of saturation effects in two-photon microscopy: artifacts in fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become an increasingly important measurement tool for biological and biomedical investigations, with the capability to assay molecular dynamics and interactions both in vitro and within living cells. Information recovery in FCS requires an accurate characterization and calibration of the observation volume. A number of recent reports have demonstrated that the calibration of the observation volume is excitation power dependent, a complication that arises due to excitation saturation. While quantitative models are now available to account for these volume variations, many researchers attempt to avoid saturation issues by working with low nonsaturating excitation intensities. For two-photon excited fluorescence, this is typically thought to be achievable by working with excitation powers for which the total measured fluorescence signal maintains its quadratic dependence on excitation intensity. We demonstrate that observing only the power dependence of the fluorescence intensity will tend to underestimate the importance of saturation, and explain these findings in terms of basic physical models.     

Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy‐to‐apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17‐day‐old laboratory Wistar rat embryo. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Morphometric characterization of murine articular cartilage—Novel application of confocal laser scanning microscopy

A new technique for characterization of the three-dimensional morphology of murine articular cartilage is proposed. The technique consists of a novel application of confocal laser scanning microscopy (CLSM), where the objective was to develop and validate it for cartilage measurements in murine joints. Murine models are used in arthritis research, because they are well-described for manipulating the disease pathophysiology, facilitating our understanding of the disease, and identifying new targets for therapy. A calibration and reproducibility study was carried out to provide a consistent testing methodology for quantification of murine joints. The proximal tibial condyles from male C57BL/6 mice were scanned using a CLS microscope with an isotropic voxel size of 5.8 μm. Measurements and analyses were repeated three times on different days, and in a second step the analysis was repeated three times for a single measurement. Calculation of precision errors (coefficient of variation) for cartilage thickness and volume was made. The bias of the system was estimated through comparison with histology. This technique showed good precision, with errors in the repeated analysis ranging from 0.63% (lateral thickness) to 3.48% (medial volume). The repeated analysis alone was robust, with intraclass correlations for the different compartments between 0.918 and 0.991. Measurement bias was corrected by scaling the confocal images to 32% of their width to match histology. CLSM provided a fast and reproducible technique for gathering 3D image data of murine cartilage and will be a valuable tool in understanding the efficacy of arthritis treatments in murine models. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc.     

Low axial drift stage and temperature controlled liquid cell for z-scan fluorescence correlation spectroscopy in an inverted confocal geometry

A recent iteration of fluorescence correlation spectroscopy (FCS), z-scan FCS, has drawn attention for its elegant solution to the problem of quantitative sample positioning when investigating two-dimensional systems while simultaneously providing an excellent method for extracting calibration-free diffusion coefficients. Unfortunately, the measurement of planar systems using (FCS and) z-scan FCS still requires extremely mechanically stable sample positioning, relative to a microscope objective. As axial sample position serves as the inherent length calibration, instabilities in sample position will affect measured diffusion coefficients. Here, we detail the design and function of a highly stable and mechanically simple inverted microscope stage that includes a temperature controlled liquid cell. The stage and sample cell are ideally suited to planar membrane investigations, but generally amenable to any quantitative microscopy that requires low drift and excellent axial and lateral stability. In the present work we evaluate the performance of our custom stage system and compare it with the stock microscope stage and typical sample sealing and holding methods.     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Optimizing the performance of confocal point scanning laser microscopes over the full field of view

To examine many of the imaging capabilities of confocal scanning laser microscopes rapidly and reliably over the whole field of view three simple, easily prepared specimens are required: a mirror positioned on a carefully measured shallow gradient, a film of highly fluorescent material and a rectangular grid with a readily defined centre. Using these specimens the adjustment of any combination of confocal scanning laser visualization system and light microscope can be examined throughout the field of view. The effects of misalignment of the various subcomponents of a confocal scanning laser microscope on both the axial spread function of a plane and the shading pattern over the image field are described. Finally, where the design of the confocal optics permits, the three specimens can be used to facilitate the alignment of the various components to the optimal level achievable.     

Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier

A novel fiber-optic confocal approach for ultrahigh depth-resolution (相似文献   

Practical limits of resolution in confocal and non-linear microscopy

Calculated and measured resolution figures are presented for confocal microscopes with different pinhole sizes and for nonlinear (2-photon and second harmonic) microscopes. A modest degree of super-resolution is predicted for a confocal microscope but in practice this is not achievable and confocal fluorescence gives little resolution improvement over widefield. However, practical non-linear microscopes do approach their theoretical resolution and therefore show no resolution disadvantage relative to confocal microscopes in spite of the longer excitation wavelength.     

Three‐dimensional imaging of porous media using confocal laser scanning microscopy

In the last decade, imaging techniques capable of reconstructing three‐dimensional (3‐D) pore‐scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO₂ storage potential. CLSM has a unique capability of producing 3‐D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z‐dimension) that can be imaged in porous materials. In this study, we introduce a ‘grind and slice’ technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3‐D confocal volumetric data into pores and grains. Finally, we use the resulting 3‐D pore‐scale binarized confocal data obtained to quantitatively determine petrophysical pore‐scale properties such as total porosity, macro‐ and microporosity and single‐phase permeability using lattice Boltzmann (LB) simulations, validated by experiments.