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.     

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.     

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.     

Demonstration of high lateral resolution in laser confocal microscopy using annular and radially polarized light

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.     

二维扫描共焦显微镜的研究

从光学系统、机械扫描、光电转换、数据采集、计算机控制、三维重建等几大方面详细地介绍了研制的二维扫描共焦显微镜 ,并对其应用前景进行了预测。     

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.     

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。     

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.     

Axial intensity in a fiber-optic confocal microscope

1Introduction Inthefieldofendoscopy,aminiaturized high resolutionimagingsystemscouldbeespe ciallyhelpfulforanumberofmedicalproblems,suchasthedeterminationoftumormarginsdur ingaminimallyinvasiveoperation,thescreening oflargetissuesections,etc.Theconfocalmicro scope[14]hasagoodprospectinendoscopy,due toitsopticalsectioningpropertyandhighreso lution.Atpresent,alotofresearchteamsinEu ropeandUSA.areworkingataclinicalendo scopewithconfocalmicroscope,whichcanafford anewandeffectivemethodtodiagnose.…     

基于区域划分的红外超分辨率重建

提出了红外超分辨率重建系统以获取高分辨率红外数据。首先,根据红外图像获取过程建立了数学模型,讨论了降采样、模糊、运动以及高斯噪声对红外系统的影响;在非退化特征提取的基础上提出了基于特征的亚像素配准算法,其根据所得到的非退化特征应用归一化均方根误差来估计两帧之间的亚像素位移。然后,分析了传统全变分因子在高分辨重建时的不足并对其进行改进;利用区域划分将图像划分为平滑区域和细节区域,并根据区域的不同情况自适应全变分因子,从而使细节区域不至于过平滑。最后,利用MM(Majorization Minimixation)算法对合成的低分辨率红外图像和真实红外图像进行了超锐度重建。与同类相关算法的比较实验显示:所提算法亚像素配准最大误差为0.09pixel,重建后的红外图像质量优于其他同类算法。所提算法可以对低分辨红外图像序列进行有效重建,具有配准精度高、重建图像细节丰富等特点,可应用于各种红外成像系统。     

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.     

Axial resolution of confocal Raman microscopes: Gaussian beam theory and practice

A straightforward and transparent model, based on Gaussian beam optics, for the axial r ₀ resolution of a confocal microscope is presented. A confocal Raman microscope was used to determine the axial confocality in practice. The axial response of a thin planar object was measured for three different objectives, two pinhole sizes and a slit. The results show that, in the case of a confocal configuration, the response calculated with the model provides a good prediction of the axial resolution of the confocal microscope.     

基于超分辨率重建的亚像素图像配准

针对低分辨率图像在配准过程中精度较低的问题,提出了一种基于超分辨率重建的亚像素图像配准方法。首先,对具有1至9像素位移的图像序列进行10倍降采样,获取具有0.1至0.9亚像素位移的图像序列。然后,根据图像的获取过程建立数学模型,以Bayes理论为基础,使用最大后验概率法(MAP)对亚像素位移低分辨率图像进行超分辨率重建,获取高分辨率图像。最后,使用具有亚像素配准精度的扩展相位相关法对图像进行配准。配准实验与噪声实验表明,所提方法的最大配准误差为0.03pixel,能实现对低分辨率图像的亚像素级配准,具有配准精度高、噪声抗干扰能力强等特点,可同时满足可见光图像与红外图像的高精度配准要求。     

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.     

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。     

稀疏表示下的噪声图像超分辨率重构

为了能够完成噪声图像的超分辨率重构,提出了一种基于稀疏表示的噪声图像超分辨率重构方法,可以同时完成图像去噪和超分辨率重构。首先,对样本图像和低分辨率图像进行块划分,建立样本库。其次,建立图像退化模型,采用相似样本加权平均的方式对输出的高分辨率图像块进行表示。根据输入的低分辨率图像块,计算样本块与输出的高分辨率图像块之间的相似性。提出了一种相似性描述方法,能够很好地解决噪声带来的影响。然后,采用相似性对稀疏编码优化模型进行惩罚,提出一种权值求解模型。模型可以自适应的搜索相似样本块而不需要预先设定相似块的个数。最后,求解权值,根据权值和样本块重构高分辨率图像块,并重构高分辨率图像。实验结果表明:所提出的方法较其它常见超分辨率算法的峰值信噪比可提高0.5dB左右,重构的图像细节更丰富,去噪效果更好,更适合实际应用。     

Theoretical versus experimental resolution in optical microscopy

The aim of this article is to compare experimental resolution under different conditions with theoretical resolution predicted using electromagnetic diffraction theory. Imaging properties of fluorescent beads of three different diameters (0.1 microm, 0.2 microm, and 0.5 microm) as well as imaging properties of four different fluorescence-stained DNA targets (ABL gene, BCR gene, centromere 6, and centromere 17) are studied. It is shown how the dependence of the resolution on object size varies with wavelength (520 nm versus 580 nm), type of microscopy (wide-field, confocal using Nipkow disk, confocal laser scanning) and basic image processing steps (median and gaussian filters). Furthermore, specimen influence on the resolution was studied (the influence of embedding medium, coverglass thickness, and depth below the coverglass). Both lateral and axial resolutions are presented. The results clearly show that real objects are far from being points and that experimental resolution is often much worse than the theoretical one. Although the article concentrates on fluorescence imaging using high NA objectives, similar dependence can also be expected for other optical arrangements.     

Increasing microscopy resolution with photobleaching and intensity cumulant analysis

Super‐resolution fluorescence microscopy and its applications for analysis of biological structures are evolving rapidly field. A number of approaches aimed at overcoming the fundamental limit imposed by diffraction have been proposed in recent years. Here we present a modification of super‐resolution optical fluctuation imaging (SOFI), a technique based on spatio‐temporal evaluation of the optical signal from independently fluctuating emitters. Instead of rapid, reversible photoswitching, photobleaching is used to produce irreversible transitions between emitting and nonemitting states of the fluorochrome molecules. Simulated images are used to demonstrate that, in the absence of noise, the proposed SOFI modification increases the efficiency of transfer of high spatial frequencies in a fluorescence microscope. Correspondingly, a decrease of the point spread function (PSF) width is obtained. Moreover, the modified SOFI algorithm is capable of resolving point emitters in the presence of simulated noise. Using real biological images we demonstrate that an increase of resolution is obtained in 2D optical sections through densely packed chromatin in cell nuclei and lamin layer at the nuclear envelope. Finally, the approach is extended to 3D wide‐field microscopy, allowing reduction of out‐of‐focus image blurring. Microsc. Res. Tech. 78:958–968, 2015. © 2015 Wiley Periodicals, Inc.     

Single-lens theta microscopy — a new implementation of confocal theta microscopy

A new microscopical technique based on the principle of confocal theta microscopy (Stelzer, E.H.K., Lindek, S. & Pick, R. (1996) Konfokales Mikroskop . German Patent Office DE 43 26 473 (filed 6.8.1993, granted 6.12.1996)) is described. It uses a single objective lens in combination with a mirror unit to achieve the theta configuration that leads to axial and volume resolution improvements. In this paper we present technical details of possible microscopical set-ups, and we discuss different versions of mirror units.     

Fluorescence saturation in confocal microscopy

The effects of fluorescence saturation on imaging in confocal microscopy have been studied. To include saturation it was necessary to deviate from the widely assumed linear relationship between the fluorescence and the illumination intensity. The lateral response for a point-like object, as well as the optical sectioning power, decreases depending on the degree of saturation. For very high illumination intensities the response for a saturated point object approached that of a conventional fluorescence microscope in which the fluorescence was not saturated. The decrease in the axial confocal response has been confirmed qualitatively by experiment.