Axial super resolution topography of focal adhesion by confocal microscopy

The protein organization within focal adhesions has been studied by state‐of‐the‐art super resolution methods because of its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation, limiting their application. In this report we present a phasor‐based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that with about 4,000 photon counts collected along a z‐scan, axial localization precision close to 10 nm is achievable. We show that, with within 10 nm, the axial location of paxillin, FAK, and talin is similar at focal adhesion sites, while F‐actin shows a sharp increase in height towards the cell center. We further demonstrated the live imaging capability of this method. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials. Microsc. Res. Tech., 76:1070–1078, 2013. © 2013 Wiley Periodicals, Inc.     

Axial resolution of confocal fluorescence microscopy

A simple analytic expression is given for the axial resolution of a confocal fluorescence microscope. The expression, which is based on the spatial frequency cut-off criterion of resolution, is valid for high aperture optics and arbitrary fluorescence wavelength.     

激光共焦扫描显微镜及其应用

介绍了共焦激光显微镜的基本光路、成像原理、关键技术及应用。     

激光共焦高速扫描显微成像的高帧速重构算法

针对激光共焦扫描显微镜的往复式逐行扫描成像方式带来的帧图像数据分割难的问题,在分析系统扫描方式、振镜的实际运动方式与理论运动方式差异的基础上,利用相邻两帧图像相似性大的特点,提出了一套完整的高帧速重构算法。该算法通过连续帧特征区域差分的方式实现了一维信号序列的自适应分割,即实现了对一维信号序列进行动态排列及分割成二维阵列图像数据,从而重构出多帧高精度图像。实验表明,该算法的成像误差低于1.6%,适用于成像速度高达300帧/s的激光共焦扫描显微成像。     

Axial resolution of confocal microscopes with parallel-beam detection

We present a simple theory for the evaluation of the axial resolution of a confocal scanning microscope with parallel-beam detection. The results demonstrate that, in certain cases, the collection efficiency is low compared with a conventional confocal microscope, but the axial resolution may be further improved.     

The experimental effect of detector size on confocal lateral resolution

The lateral resolution of a scanning-spot microscope has been measured as the product of the pin-hole size and lens numerical aperture of the detector approaches the confocal limit. The resolution follows an earlier prediction, improving approximately 30% in going from that of a conventional scanning-spot or broad-viewing-area microscope to that of a fully confocal microscope. The discrepancy between the data and the theoretical curve can be attributed to lens apodization arising from the use of multi-element thick lenses and a non-ideal, truncated Gaussian beam profile.     

Automated compensation of light attenuation in confocal microscopy by exact histogram specification

Confocal laser scanning microscopy (CLSM) enables us to capture images representing optical sections on the volume of a specimen. The images acquired from different layers have a different contrast: the images obtained from the deeper layers of the specimen will have a lower contrast with respect to the images obtained from the topmost layers. The main reasons responsible for the effects described above are light absorption and scattering by the atoms and molecules contained in the volume through which the light passes. Also light attenuation can be caused by the inclination of the observed surface. In the case of the surfaces that have a steep inclination, the reflected light will have a different direction than the one of the detector. We propose a technique of digital image processing that can be used to compensate the effects of light attenuation based on histogram operations. We process the image series obtained by CLSM by exact histogram specification and equalization. In this case, a strict ordering among pixels must be induced in order to achieve the exact histogram modeling. The processed images will end up having exactly the specified histogram and not a histogram with a shape that just resembles to the specified one, as in the case of classical histogram specification algorithms. Experimental results and theoretical aspects of the induced ordering are discussed, as well as a comparison between several histogram modeling techniques with respect to the processing of image series obtained by confocal microscopy. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

The effect of detector size on the signal-to-noise ratio in confocal polarized light microscopy

We introduce a signal-to-noise ratio in an attempt to suggest an optimum pinhole size for confocal polarized light microscopes. We find that pinhole sizes which are typically 60% greater than those used in nonpolarized light confocal microscopy are appropriate.     

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross‐talk between adjacent imaging channels. We demonstrate that a time‐multiplexed non‐linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single‐photon confocal system. The background becomes irrelevant for thin (< 15 µm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

Advances in laser sources for confocal and multiphoton microscopy

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.     

High temporal and spatial resolution studies of bone cells using real-time confocal reflection microscopy

Chick and rat bone-derived cells were mounted in sealed coverslip-covered chambers; individual osteoclasts (but also osteoblasts) were selected and studied at 37°C using three different types of high-speed scanning confocal microscopes: (1) A Noran Tandem Scanning Microscope (TSM) was used with a low light level, cooled CCD camera for image transfer to a Noran TN8502 frame store-based image analysing computer to make time lapse movie sequences using 0.1 s exposure periods, thus losing some of the advantage of the high frame rate of the TSM. Rapid focus adjustment using computer controlled piezo drivers permitted two or more focus planes to be imaged sequentially: thus (with additional light-source shuttering) the reflection confocal image could be alternated with the phase contrast image at a different focus. Individual cells were followed for up to 5 days, suggesting no significant irradiation problem. (2) Exceptional temporal and spatial resolution is available in video rate laser confocal scanning microscopes (VRCSLMs). We used the Noran Odyssey unitary beam VRCSLM with an argon ion laser at 488 nm and acousto-optic deflection (AOD) on the line axis: this instrument is truly and adjustably confocal in the reflection mode. (3) We also used the Lasertec 1LM11 line scan instrument, with an He-Ne laser at 633 nm, and AOD for the frame scan. We discuss the technical problems and merits of the different approaches. The VRCSLMs documented rapid, real-time oscillatory motion: all the methods used show rapid net movement of organelles within bone cells. The interference reflection mode gives particularly strong contrasts in confocal instruments. Phase contrast and other interference methods used in the microscopy of living cells can be used simultaneously in the TSM.     

激光差动共焦透镜中心厚度测量系统的研制

基于高精度光学共焦定位技术研制了一种全新的非接触透镜中心厚度测量系统,该系统利用差动共焦技术的高轴向层析特性和轴向响应曲线的绝对零点对被测透镜的前表面顶点和后表面顶点分别进行精密瞄准定位;同时,利用激光干涉仪获得透镜前、后表面顶点的位置坐标;然后通过光线追迹算法计算透镜中心厚度,进而实现了透镜中心厚度的高精度非接触测量。实验结果表明,该系统测量精度高,测量标准差小于1μm,满足透镜中心厚度测量的精度要求。     

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy

Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.     

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.     

Ultrathin fluorescent layers for monitoring the axial resolution in confocal and two-photon fluorescence microscopy

Monomolecular films of polymerized dimethyl-bis[pentacosadiinoic-oxyethyl] ammonium bromide (EDIPAB) provide one- and two-photon excited fluorescence that is sufficiently high to quantify the axial resolution of 3-D fluorescence microscopes. When scanned along the optical axis, the fluorescence of these layers is bright enough to allow online observation of the axial response of these microscopes, thus facilitating alignment and fluorescence throughput control. The layers can be used for directly measuring and monitoring the axial response of 4Pi-confocal microscopes, as well as for their initial alignment and phase adjustment. The proposed technique has the potential to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Coverslips with EDIPAB-layers can be used as substrates for the cultivation of cells.     

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.     

The role of specimen-induced spherical aberration in confocal microscopy

We present an overview of recent theories for describing specimen-induced spherical aberration in confocal microscopy. One of these theories is used to compute numerically the role of spherical aberration in general confocal, and especially in biological confocal, microscopy for a variety of three-layer specimen structures. In particular, we study the effect of specimen-induced spherical aberration on the maximum value of the overall confocal point spread function, the accompanying focal shift and the size of the optical probe in both fluorescence and brightfield confocal microscopy.     

Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz

The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small (< 1 μm), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (> 10 μm) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.