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1.
We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.  相似文献   

2.
浅谈共聚焦显微技术   总被引:1,自引:1,他引:0  
陈木旺 《光学仪器》2013,35(1):44-47
共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。  相似文献   

3.
The exposure of fluorophores to intense illumination in a microscope often results in photobleaching and phototoxicity, thus constituting a major limiting factor in time lapse live cell or single molecule imaging. Laser scanning confocal microscopes are particularly prone to this problem, inasmuch as they require high irradiances to compensate for the inherently low duty cycle of point scanning systems. In the attempt to maintain adequate speed and signal-to-noise ratios, the fluorophores are often driven into saturation, thereby generating a nonlinear response. One approach for reducing photodegradation in the laser scanning confocal microscope is represented by controlled light exposure microscopy, introduced by Manders and colleagues. The strategy is to reduce the illumination intensity in both background areas (devoid of information) as well as in bright foreground regions, for which an adequate signal-to-noise ratio can be achieved with lower excitation levels than those required for the less intense foreground pixels/voxels. Such a variable illumination scheme can also be exploited in widefield microscopes that employ lower irradiance but higher illumination duty cycles. We report here on the adaptation of the controlled light exposure microscopy principle to the programmable array microscope, which achieves optical sectioning by use of a spatial light modulator (SLM) in an image plane as a programmable mask for illumination and conjugate (and nonconjugate) detection. By incorporating the basic controlled light exposure microscopy concept for minimizing exposure, we have obtained a reduction in the rate of photobleaching of up to ~5-fold, while maintaining an image quality comparable to regular imaging with the programmable array microscope.  相似文献   

4.
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.  相似文献   

5.
针对当前宽厚板板形在线智能化检测的需求,设计一种单激光器多相机的宽厚板板形测量系统,提出一种基于唯一激光平面的多相机坐标系姿态校准标定方法。板形测量系统由分别位于宽厚板矫正机前后的两个相同的子系统组成,子系统采用多相机测量视野拼接的方式,实现宽厚板板形的检测;利用相邻相机公共视野中同一姿态标定板世界坐标系的唯一性,将多个相机的坐标系映射在基准坐标系下,根据实际激光平面的唯一性将多个相机坐标系统一为同一姿态,并通过统一坐标后相邻相机点云数据的位置关系,实现多相机测量数据的快速拼接,完成多相机结构光测量系统坐标系的标定。试验结果表明,该方法准确有效,为大视场结构光三维测量提供了一种有效的测量标定方法。  相似文献   

6.
占栋  肖建 《仪器仪表学报》2015,36(9):2030-2036
多摄像机视觉测量系统中不同视觉传感器空间分布广,无公共视角,现场标定十分困难。针对多摄像机标定问题,研究了一种基于线结构光参考平面的灵活标定方法。标定过程中,以空间中同时覆盖相邻摄像机视角的结构光平面作为标定参考基准,在不同摄像机视角中,自由移动平面靶标多次,确保每次移动后靶标与结构光相交,并能在各自摄像机中清晰成像,摄像机拍摄靶标图像。提取靶标图像中角点坐标、激光光条特征点坐标。借助靶标平面与摄像机坐标系外部参数矩阵,求解激光光条特征点在对应摄像机坐标系中坐标。通过结构光基准平面内,不同摄像机坐标系中至少3组非共线特征点坐标信息,求解相邻摄像机外部参数。分别进行标定试验和精度验证试验,试验结果表明该方法切实可行。  相似文献   

7.
基于三点透视模型的线结构光系统标定方法   总被引:1,自引:0,他引:1  
针对现有线结构光传感系统标定过程中对设备要求高、标定过程繁琐等问题,提出一种基于三点透视模型的快速标定方法。引入一个可自由移动的平面靶标,靶标上只需要共线、且相互位置确定的三个特征点,利用共线三点建立三点透视数学模型,根据三个特征点以及光条纹在摄像机像面的成像信息,就可以获取光平面上标定点在摄像机坐标系下的坐标。平面靶标在视觉范围内任意移动几个位置,得到光平面上多个标定点坐标,从而确定光平面方程。实验证明,该方法平均相对测量误差小于0.8%。该方法不需要昂贵的辅助调整设备,也不需要求解坐标系之间的转换矩阵,简单、快速,适合现场标定。  相似文献   

8.
In widefield fluorescence microscopy, images from all but very flat samples suffer from fluorescence emission from layers above or below the focal plane of the objective lens. Structured illumination microscopy provides an elegant approach to eliminate this unwanted image contribution. To this end a line grid is projected onto the sample and phase images are taken at different positions of the line grid. Using suitable algorithms ‘quasi‐confocal images’ can be derived from a given number of such phase‐images. Here, we present an alternative structured illumination microscopy approach, which employs two‐dimensional patterns instead of a one‐dimensional one. While in one‐dimensional structured illumination microscopy the patterns are shifted orthogonally to the pattern orientation, in our two‐dimensional approach it is shifted at a single, pattern‐dependent angle, yet it already achieves an isotropic power spectral density with this unidirectional shift, which otherwise would require a combination of pattern‐shift and ‐rotation. Moreover, our two‐dimensional approach also yields a better signal‐to‐noise ratio in the evaluated image.  相似文献   

9.
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.  相似文献   

10.
We have developed a non-optically probing near-field microscope with illumination of total internal reflection. Because the illumination light does not pass through the specimens, it is possible to observe thick specimens or highly absorptive materials. It reduces the background noise because the decay length of the evanescent wave is a few hundred nanometres. We found that although in the total internal reflection illumination system the light passed through the photosensitive film and illuminated the specimen, it did not affect the photosensitive film severely and did not limit the resolution. The imaging properties of reflection illumination and transmission illumination are analysed using a finite-differential time domain method.  相似文献   

11.
In recent years, high-resolution microscopy using structured illumination has been practically applied for fluorescent bio-imaging. However, there is a large amount of speckle noise in reflected- and scattered-light images, because structured illumination is typically generated by laser-beam interference. Hence, this high-resolution imaging technique cannot be effectively used in industrial applications. In this study, we attempted to generate structured illumination using two-beam interference of low-coherence light for high-resolution and low-speckle imaging. First, we constructed an optical system consisting of a Michelson interferometer configured in such a manner that it achieved zero optical path-length difference and allowed the interference fringes to be manipulated. Then, we confirmed that the generated structured illumination width corresponded to the coherence length of the light source. As a final result of the resolution improvement experiment, the narrow sample pitch of 0.4 μm was successfully resolved beyond the diffraction limit of 0.74 μm with relatively less speckle noise.  相似文献   

12.
The imaging characteristics of a confocal scanning light microscope (CSLM) with high aperture, immersion type, lenses (N.A. = 1·3) are investigated. In the confocal arrangement the images of the illumination and detector pinholes are made to coincide in a common point, through which the object is scanned mechanically. Results show that for point objects the theoretically expected improved response by a factor of 1·4 in comparison with standard microscopy can indeed be realized. Low side lobe intensity and absence of glare permits the imaging at high resolution of weak details close to strong features. A further improvement by a factor of 1·25 in point resolution in CSLM is found after apodization with an annular aperture. Due to the scanning approach all possibilities of electronic image processing become available in light microscopy.  相似文献   

13.
The fine structure of the in-situ rabbit crystalline ocular lens from the ex-vivo rabbit eye was observed with a confocal scanning laser microscope in the scattered light mode. The images were observed through the full thickness of the cornea and aqueous humour to a depth of 50 μm in the anterior ocular lens. The following structures were observed from optical sections of the ocular lens: two concentric regions of the lens capsule, epithelial cells, lens sutures, and surface and interior regions of individual lenticular fibres. The observed lateral resolution of the microscope objective was degraded by imaging across thick (millimetre) structures. This study shows the feasibility of obtaining high-contrast images of transparent objects across 1.7 mm of ocular tissue (cornea and aqueous humour) using confocal light microscopy.  相似文献   

14.
Two‐photon fluorescence microscopy and confocal reflectance microscopy were compared to detect intracellular gold nanorods in rat basophilic leukaemia cells. The two‐photon photoluminescence images of gold nanorods were acquired by an 800 nm fs laser with the power of milliwatts. The advantages of the obtained two‐photon photoluminescence images are high spatial resolution and reduced background. However, a remarkable photothermal effect on cells was seen after 30 times continuous scanning of the femto‐second laser, potentially affecting the subcellular localization pattern of the nanorods. In the case of confocal reflectance microscopy the images of gold nanorods can be obtained with the power of light source as low as microwatts, thus avoiding the photothermal effect, but the resolution of such images is reduced. We have noted that confocal reflectance images of cellular gold nanorods achieved with 50 μW 800 nm fs have a relatively poor resolution, whereas the 50 μW 488 nm CW laser can acquire reasonably satisfactory 3D reflectance images with improved resolution because of its shorter wavelength. Therefore, confocal reflectance microscopy may also be a suitable means to image intracellular gold nanorods with the advantage of reduced photothermal effect.  相似文献   

15.
We describe the development of a beam scanning microscope that can perform optical sectioning based on the principle of confocal microscopy. The scanning is performed by a laser beam diffracted from a dynamic binary hologram implemented using a liquid crystal spatial light modulator. Using the proposed scanning mechanism, unlike the conventional confocal microscopes, scanning over a two-dimensional area of the sample can be obtained without the use of a pair of galvo mirror scanners. The proposed microscope has a number of advantages, such as superior frame to frame repeatability, simpler optical arrangement, increased pixel dwell time relative to the time between two pixels, illumination of only the sample points without pulsing the laser, and absolute control over the amplitude and phase of the illumination beam on a pixel to pixel basis. The proposed microscope can be particularly useful for applications requiring very long exposure time or very large working distance objective lenses. In this paper we present experimental implementation of the setup using a nematic liquid crystal spatial light modulator and proof-of-concept experimental results.  相似文献   

16.
Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm?2 and about 300 J cm?2 for native cells (corresponding to about 250?3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi‐ or total internal reflection illumination) and decreased to values between 50 J cm?2 and less than 1 J cm?2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high‐resolution wide field or laser scanning microscopy of single cells, typically 10?20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super‐resolution microscopy based on single molecule detection.  相似文献   

17.
由于受到光学衍射的限制,均匀照明宽视场荧光显微术和激光共焦扫描显微术的分辨率约为200~300nm。近年来受激发射损耗显微术在突破衍射极限以及应用方面取得许多令人瞩目的成果。本文简要介绍受激发射损耗显微术的原理、方法及其在生物医学上的应用。  相似文献   

18.
Activated sludge flocs are complex consortia of various micro-organisms. The community structures of samples taken from municipal sewage treatment plants were characterized using fluorescently labelled, 16S and 23S rRNA-targeted oligonucleotide probes in combination with confocal scanning laser microscopy (CSLM). In comparison with conventional epifluorescence microscopy, CSLM considerably improved the capability to visualize directly the spatial distribution of defined bacterial populations inside the sludge flocs. Analyses could be performed at high resolution undisturbed by problems such as autofluorescence or limited spatial resolution in thick samples. In addition, CSLM was used to analyse some structural properties of paraformaldehyde-fixed activated sludge flocs, such as floc size and homogeneity. Typical floc sizes were found to be in the range between 5 and 50 μm. Whereas most of the flocs were completely colonized by bacteria, there were also examples of flocs containing gas bubbles or particles in the interior.  相似文献   

19.
The authors present the experimental result of improved lateral resolution in laser confocal microscopy (LCM) by using annular and radially polarized light as the input illumination of an existing LCM. The authors examined the lateral resolution of the LCM by imaging a single fluorescent bead and measuring the lateral width of the single bead profile appearing in the optical image. Compared to no aperture and linearly polarized light, the central peak of the single bead profile narrowed by ∼40%, being as small as 122 nm in full width at half maximum using 405 nm laser excitation in a reflection imaging. In addition, the authors showed that radial polarization helps to preserve the circular shape of the single bead profile whereas linearly polarized light tends to induce an elongation along the polarization direction. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.  相似文献   

20.
We review the origins of optical sectioning in fluorescence microscopy in terms of the structure of the illumination used to generate the fluorescence within the specimen. We note that the conventional microscope using essentially uniform illumination does not exhibit optical sectioning whereas the confocal microscope using point (many spatial frequencies) illumination does. We show that the optical sectioning strength of a confocal microscope is not optimal and discuss the advantages of using a single spatial frequency for the structure of the illumination and the detection. In this case the optical sectioning strength is shown to be up to 25% narrower than in the ideal confocal case.  相似文献   

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