Deconvolution Combined with Digital Colocalization Analysis to Study the Spatial Distribution of Tight and Adherens Junction Proteins

The relative spatial distribution of proteins was investigated with immunofluorescent methods by confocal laser scanning microscopy and digital image restoration. For confocal data sets recorded with a voxel dimension of 50 × 50 × 150 nm noise and blur can be decreased and the resolution in the z-axis increased by applying the maximum likelihood estimation algorithm of the Huygens software. This approach was successfully applied to the study of tight and adherens junctions in relation to the actin cytoskeleton in Madin Darby Canine Kidney cells. Colocalization analysis was done for pairs of probes using a histogram-based method. F-actin, occludin, zonula occludens 1, and E-cadherin were included in the study. Double-labeled preparations were used. The combination of deconvolution with the colocalization of confocal data sets offers a powerful tool to investigate the spatial arrangement of proteins.     

Penetration pathways of fluorescent dyes in human hair fibres investigated by scanning near-field optical microscopy

Thin cross-sections of human hairs were investigated by scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) after penetration of a fluorescent dye. The same samples were measured with both techniques to compare the observed structures. The images obtained from the two methods show nearly identical structures representing pathways of the dye molecules in hairs. The SNOM images provide a higher resolution than the CLSM images. Therefore, SNOM is believed to be a suitable method for investigations at a resolution of 100 nm on penetration pathways of fluorescent dyes such as the cell membrane complex pathway in cross-sections of hairs.     

Detection of endolithic spatial distribution in marble stone

The penetration of endolithic microorganisms, which develop to depths of several millimetres or even centimetres into the stone, and the diffusion of their extracellular substances speeds up the stone deterioration process. The aim of this study was to investigate, using a confocal laser scanning microscopy with a double‐staining, a marble rock sample by observing the endolithic spatial distribution and quantifying the volume they occupied within the stone, in order to understand the real impact of these microorganisms on the conservation of stone monuments. Often the only factors taken into account by biodeterioration studies regarding endolithic microorganisms, are spread and depth of penetration. Despite the knowledge of three‐dimensional spatial distribution and quantification of volume, it is indispensable to understand the real damage caused by endolithic microorganisms to stone monuments. In this work, we analyze a marble rock sample using a confocal laser scanning microscopy stained with propidium iodide and Concavalin‐A conjugate with the fluorophore Alexa Fluor 488, comparing these results with other techniques (SEM microscope, microphotographs of polished cross‐sections and thin‐section, PAS staining methods), An image analysis approach has also been applied. The use of confocal laser scanning microscopy with double staining shows clear evidence of the presence of endolithic microorganisms (cyanobacteria and fungi) as well as the extracellular polymeric substance matrix in a three‐dimensional architecture as part of the rock sample, this technique, therefore, seems very useful when applied to restoration interventions on stone monuments when endolithic growth is suspected.     

Configurations of the Re‐scan Confocal Microscope (RCM) for biomedical applications

The new high‐sensitive and high‐resolution technique, Re‐scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re‐scan detection unit. The re‐scan unit includes a pair of re‐scanning mirrors that project the emission light onto a camera in a scanning manner. The signal‐to‐noise ratio of Re‐scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re‐scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re‐scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re‐scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re‐scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio‐metric (e.g. pH and intracellular Ca²⁺ measurements) and FRAP imaging.     

A tilting device for three-dimensional microscopy: Application to in situ imaging of interphase cell nuclei

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the focal plane (x-y plane). This is true of conventional as well as confocal microscopes. For quantitative microscopy, for instance studies of the three-dimensional (3-D) organization of chromosomes in human interphase cell nuclei, the 3-D image must be reconstructed by a point spread function or an optical transfer function with careful consideration of the properties of the imaging system. To alleviate the reconstruction problem, a tilting device was developed so that several data sets of the same cell nucleus under different views could be registered. The 3-D information was obtained from a series of optical sections with a Zeiss transmission light microscope Axiomat using a stage with a computer-controlled stepping motor for movement in the z-axis. The tilting device on the Axiomat stage could turn a cell nucleus through any desired angle and also provide movement in the x-y direction. The technique was applied to 3-D imaging of human lymphocyte cell nuclei, which were labelled by in situ hybridization with the DNA probe pUC 1.77 (mainly specific for chromosome 1). For each nucleus, 3-D data sets were registered at viewing angles of 0°, 90° and 180°; the volumes and positions of the labelled regions (spots) were calculated. The results also confirm that, in principle, any angle of a 2p geometry can be fixed for data acquisition with a high reproducibility. This indicates the feasibility of axiotomographical microscopy of cell nuclei.     

Enhanced scanning control in a confocal scanning laser microscope

A fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user-selected closed curves. The scanning unit uses two galvanometer-mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time-resolved recordings within the millisecond range can be performed. Repetition rates up to >1 kHz for line scanning and curve scanning, and >100 kHz for point scanning are obtainable. This allows time-resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal-to-noise ratio obtainable with a point scanning confocal microscope.     

Practical limitations of superresolution imaging due to conventional sample preparation revealed by a direct comparison of CLSM,SIM and dSTORM

We evaluate the suitability of conventional sample preparation and labelling methods for two superresolution techniques, structured illumination microscopy and direct stochastic optical reconstruction microscopy, by a comparison to established confocal laser scanning microscopy. We show that SIM is compatible with standard fixation procedures and immunofluorescence labelling protocols and improves resolution by a factor of two compared to confocal laser scanning microscopy. With direct stochastic optical reconstruction microscopy, fluorophores can theoretically be localized with much higher precision. However, in practice, with indirect immunofluorescence labelling density can be insufficient due to the bulky probes to reveal biological structures with high resolution. Fine structures like single actin fibres are in fact resolved with direct stochastic optical reconstruction microscopy when using small affinity probes, but require proper adjustment of the fixation protocol. Finally, by a direct comparison of immunofluorescent and genetic labelling with fluorescent proteins, we show that target morphology in direct stochastic optical reconstruction microscopy data sets can differ significantly depending on the labelling method and the molecular environment of the target.     

Enhanced scanning control in a confocal scanning laser microscope

A fast and flexible scanning unit, allowing scanning rates of more than 1 kHz over regions identified in a specimen, has been developed and evaluated. This scanning unit replaces the original scanning unit in the Phoibos confocal scanning laser microscope and features full backward compatibility, while at the same time allowing fast and flexible scanning modes, such as point scanning, line scanning, and scanning along user-selected closed curves. The scanning unit uses two galvanometer-mounted mirrors for scanning. A standard procedure for recordings with this scanning unit would be to scan an overview image with conventional raster scanning to identify a region of interest, mark a point, a line, or a closed curve over this region, and to start the scanner. An iterating algorithm then calculates the waveforms needed by the scanner to follow the identified curves with pixel precision. With this scanning unit and its controlling software, experiments demanding time-resolved recordings within the millisecond range can be performed. Repetition rates up to > 1 kHz for line scanning and curve scanning, and > 100 kHz for point scanning are obtainable. This allows time-resolved studies of fast reactions in living tissue to be performed with the spatial resolution and signal-to-noise ratio obtainable with a point scanning confocal microscope.     

全体件表面数字化多视拼合的系统方法

给出了解决三坐标测量中多视数据拼合的系统方法。首先通过坐标转化将多视数据统一化 ,然后将空间的数据点投影至平面上形成单视数据点的三角拓扑关系 ,使散乱的数据点具有面信息。利用射线交点记数法将重叠处的点从另一视的数据集中分离出来 ,提出了将多值数据点自适应滤除的新方法 ,最后基于外接圆规则重新三角化     

Spatial calibration of structured illumination fluorescence microscopy using capillary tissue phantoms

Quantitative assessment of microvascular structure is relevant to the investigations of ischemic injury, reparative angiogenesis and tumor revascularization. In light microscopy applications, thick tissue specimens are necessary to characterize microvascular networks; however, thick tissue leads to image distortions due to out-of-focus light. Structured illumination confocal microscopy is an optical sectioning technique that improves contrast and resolution by using a grid pattern to identify the plane-of-focus within the specimen. Because structured illumination can be applied to wide-field (nonscanning) microscopes, the microcirculation can be studied by sequential intravital and confocal microscopy. To assess the application of structured illumination confocal microscopy to microvessel imaging, we studied cell-sized microspheres and fused silica microcapillary tissue phantoms. As expected, structured illumination produced highly accurate images in the lateral (X-Y) plane, but demonstrated a loss of resolution in the Z-Y plane. Because the magnitude of Z-axis distortion was variable in complex tissues, the silica microcapillaries were used as spatial calibration standards. Morphometric parameters, such as shape factor, were used to empirically optimize Z-axis software compression. We conclude that the silica microcapillaries provide a useful tissue phantom for in vitro studies as well as spatial calibration standard for in vivo morphometry of the microcirculation.     

Three-dimensional optical microscopy using tilted views

The resolution of an optical microscope is considerably less in the direction of the optical axis (z) than in the x–y plane. This is true of conventional or confocal microscopes. To alleviate this problem we used multiple tilted views to supply the ‘missing data’ and thus increase the resolution in z. A special tilting stage was constructed which allowed specimens to be rotated through large angles. The relative, translation, rotation and z-spacing between data sets were determined by a novel Wiener/phase cross-correlation function. Once brought to a common coordinate system the data sets can be combined by Fourier space techniques similar to those used in X-ray crystallography. We applied this technique to metaphase chromosomes from intact embryos of Drosophila melanogaster. As determined from significant intensity in the Fourier transform, the resolution of the final reconstruction was about 0?25 μm in x and y, and 0?4 μm in z.     

Axial tomographic confocal fluorescence microscopy

By physical rotation of the sample, axial tomography enables the acquisition of otherwise inaccessible spatial information from an object. In combination with confocal microscopy, the method can fundamentally improve the effective three‐dimensional (3D) resolution. In this report we present a novel method for high resolution reconstruction of confocal axial tomographic data. The method automatically determines the relative angles of rotation, aligns the data from different rotational views and reconstructs a single high resolution 3D dataset. The reconstruction makes use of a known point spread function and is based on an unconstrained maximum likelihood deconvolution performed simultaneously from multiple (in our case three) angular views. It was applied to simulated as well as to experimental confocal datasets. The gain in resolution was quantified and the effect of choice of overrelaxation factors on the speed of convergence was investigated. A clearly improved 3D resolution was obtained by axial tomography together with reconstruction as compared with reconstruction of confocal data from only a single angular view.     

Fluorescence lifetime imaging by time-correlated single-photon counting

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.     

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.     

Measurement of co-localization of objects in dual-colour confocal images

A method to measure the degree of co-localization of objects in confocal dual-colour images has been developed. This image analysis produced two coefficients that represent the fraction of co-localizing objects in each component of a dual-channel image. The generation of test objects with a Gaussian intensity distribution, at well-defined positions in both components of dual-channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co-localization coefficients were determined before degrading the image with background, cross-talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non-specific binding and cross-reactivity of fluorescent probes, optical cross-talk and photon noise. The degraded images were restored by filtering and cross-talk correction. The co-localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co-localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.     

Alpha particle spectroscopy using FNTD and SIM super‐resolution microscopy

Structured illumination microscopy (SIM) for the imaging of alpha particle tracks in fluorescent nuclear track detectors (FNTD) was evaluated and compared to confocal laser scanning microscopy (CLSM). FNTDs were irradiated with an external alpha source and imaged using both methodologies. SIM imaging resulted in improved resolution, without increase in scan time. Alpha particle energy estimation based on the track length, direction and intensity produced results in good agreement with the expected alpha particle energy distribution. A pronounced difference was seen in the spatial scattering of alpha particles in the detectors, where SIM showed an almost 50% reduction compared to CLSM. The improved resolution of SIM allows for more detailed studies of the tracks induced by ionising particles. The combination of SIM and FNTDs for alpha radiation paves the way for affordable and fast alpha spectroscopy and dosimetry.     

基于数字微镜器件的三维轮廓测量及其性能分析

研究提出了一种基于数字微镜器件(digital micromirror device,DMD)的并行三维微-纳米轮廓测量方法.该方法将数字微镜器件与共焦测量方法相结合,用数字微镜器件及其控制器替代了传统共焦测量中的照明针孔和横向扫描机构,充分发挥DMD横向分辨率高,响应速度快,数字化以及便于计算机控制的优点,对传统共焦测量方法进行了结构上的改进,大大提高其扫描速度.一系列实验结果表明,数字微镜器件可以完全替代照明针孔,从而实现对样品表面快速,精确,大范围的三维测量.     

Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores under confocal microscopy

Ratiometric quantification of CFP/YFP FRET enables live-cell time-series detection of molecular interactions, without the need for acceptor photobleaching or specialized equipment for determining fluorescence lifetime. Although popular in widefield applications, its implementation on a confocal microscope, which would enable sub-cellular resolution, has met with limited success. Here, we characterize sources of optical variability (unique to the confocal context) that diminish the accuracy and reproducibility of ratiometric FRET determination and devise practical remedies. Remarkably, we find that the most popular configuration, which pairs an oil objective with a small pinhole aperture, results in intractable variability that could not be adequately corrected through any calibration procedure. By quantitatively comparing several imaging configurations and calibration procedures, we find that significant improvements can be achieved by combining a water objective and increased pinhole aperture with a uniform-dye calibration procedure. The combination of these methods permitted remarkably consistent quantification of sub-cellular FRET in live cells. Notably, this methodology can be readily implemented on a standard confocal instrument, and the dye calibration procedure yields a time savings over traditional live-cell calibration methods. In all, identification of key technical challenges and practical compensating solutions promise robust sub-cellular ratiometric FRET imaging under confocal microscopy.     

Imaging modes for bilateral confocal scanning microscopy

The bilateral scanning approach to confocal microscopy is characterized by the direct generation of the image on a two-dimensional (2-D) detector. This detector can be a photographic plate, a CCD detector or the human eye, the human eye permitting direct visualization of the confocal image. Unlike Nipkow-type systems, laser light sources can be used for excitation. A design called a carousel has been developed, in which the bilateral confocal scan capability can be added to an existing microscope so that rapid exchange and comparison between confocal and non-confocal imaging conditions is possible. The design permits independent adjustment of confocal sectioning properties with lateral resolutions better than, or, in the worst case equivalent to, those available in conventional microscopy. The carousel can be considered as a stationary optical path in which certain imaging conditions, such as confocality, are defined and operate on part of the imaging field. The action of the bilateral scan mirror then extends this image condition over the whole field. A number of optical arrangements for the carousel are presented which realize various forms of confocal fluorescence and reflection imaging, with point, multiple point or slit confocal detection arrangements. Through the addition of active elements to the carousel direct stereoscopic, ratio, time-resolved and other types of imaging can be achieved, with direct image formation on a CCD, eye or other 2-D detectors without the need to modify the host microscope. Depending on the photon flux available, these imaging modes can run in real-time or can use a cooled CCD at (very) low light level for image integration over an extended period.     

Imaging in the far-red with electronic light microscopy: Requirements and limitations

The acquisition of simultaneous dual confocal images with red and far-red light has both advantages (e.g. lower autofluorescence) and limitations. An understanding of these requisites is necessary to acquire high-quality images and to avoid the misinterpretation of experimental data. The poor detection of far-red light mandates a high optical transfer efficiency for the system, thus the transmittance of the objective lens and its axial and lateral chromatic aberration in the far-red are important factors for consideration. This technical note is an attempt to ‘demystify’ the process of filter set design for confocal microscopy by discussing the considerations that went into the construction of a filter set for use with the reagents cyanine 3.18 (Cy3) and cyanine 5.18 (Cy5), and thus to encourage users to look beyond the multi-purpose designs available commercially. The 568-nm laser line exciting Cy3 is at its emission maximum, which limits the collectable Cy3 fluorescence. High-transmission optical filters with sharp band pass cutoffs are thus desirable for maximum light throughput. Light path mirror efficiency rapidly degrades above 700 nm, but the loss of this portion of the Cy5 emission spectrum is acceptable since the fluorophore is very bright, and these very long wavelengths are also likely to introduce aberration. While resolution is decreased with far-red light, there is also greater penetration and less scattering, and it is thus possible to obtain high-quality images from deeper within the specimen. Although only one make and model of confocal microscope (the Bio-Rad MRC–600) is considered, similar considerations pertain to the design of filter sets for any confocal microscope that accommodates user-installed filters.