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.     

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。     

三维显微图像带约束的迭代解卷积复原算法

阐述显微镜透明厚样本成像原理和三维显微图像带约束的迭代解卷积复原算法。根据显微镜透明厚样本成像原理,对已知三维清晰图像进行退化处理,并且使用带约束的迭代解卷积算法去除退化图像中的散焦信息。试验结果表明,图像散焦信息的干扰得到有效的去除,清晰度和信噪比得到明显的改善,并且该算法可以恢复成像过程中丢失的部分频率成分,实现超分辨率复原。当迭代次数较大时,复原效果优于邻域法和线性方法。     

The quantification of fluorescent emission from biological samples using analysis of polarization

The quantification of fluorescent emission from biological specimens can only be carried out in cellular regions where the relationship between fluorophore concentration and fluorescent emission is linear. Using a confocal scanning laser microscope, we show that quantification of fluorescent emission from biological samples labelled with fluorescein and fluorescein analogues mounted in a viscous medium can be readily achieved. Where the distribution of fluorophore is highly localized, for example in cells labelled for immunofluorescence analysis, we demonstrate that analysis of fluorescence depolarization can identify regions in which fluorophore concentration exceeds the range in which the relationship to fluorescent emission is linear. We also demonstrate that, under the conditions examined, depth-dependent effects, fading and quenching are either small enough to be ignored or can be corrected for mathematically when quantifying fluorescent emission.     

The use of polarization analysis in the quantification of fluorescent emission: general principles

Fluorescent emission and fluorophore concentration are only linearly related below particular concentrations of fluorophore. Theoretically, analysis of fluorescent polarization might allow identification of situations in which local concentrations of fluorophore are above the range of linear response. Using a confocal scanning laser microscope, we demonstrate that progressive depolarization of fluorescent emission from fluorescein and fluorescein analogues occurs over the concentration range where linearity is lost. Critically, depolarization of emission is first seen at concentrations of fluorophore slightly below those at which linearity is lost. Thus, polarization analysis can be used to determine whether the local concentration of fluorophore is such that quantitative analysis can be carried out.     

基于多传感器的红外图像超分辨

利用可见光图像与红外图像之间存在的互补性和相关性,提出一种基于多传感器的红外图像超分辨方法。首先,利用自适应边缘检测算法提取红外图像边缘,并根据红外图像边缘与可见光图像对应区域的相关程度将红外图像边缘分为相关边缘和非相关边缘;然后,采用二次关系模型对相关边缘区域进行建模,通过该模型利用可见光信息估计红外图像的高频信息;最后,利用迭代反向投影法(IBP)对估计的高分辨率红外图像进行优化获得最终的高分辨率红外图像。实验结果表明,本文算法获得的小区图像,十字路口图像和道路图像的峰值信噪比(PSNR)分别比Choi算法高2.9dB,1.44dB和1.11dB。另外,利用本文算法复原的红外图像具有更好的视觉效果,更逼真、更接近于原始高分辨率图像,复原出的高分辨率红外图像无论在主观效果上还是在客观评价指标上都取得了较好的结果。     

Calibration of step heights and roughness measurements with atomic force microscopes

In this paper we present a method for the vertical calibration of a metrological atomic force microscope (AFM), which can be applied to most AFM systems with distance sensors. A thorough analysis describes the physical z-coordinate of an imaged surface as a function of the observed and uncorrected z-coordinate and the horizontal position. The three most important correction terms in a Taylor expansion of this function are identified and estimated based on series of measurements on a calibrated step height and a flat reference surface. Based on this calibration a number of step heights are calibrated by the AFM with measured values consistent with reference values, where available. Relative standard uncertainty of about 0.5% is achieved for step heights above 200 nm. For step heights below 50 nm, the standard uncertainty is about 0.5 nm. While a calibration of step heights done by AFM and interference microscopy can be compared directly as demonstrated here, this is not straightforward for roughness measurement. To asses this, the exact same area on an important applied surface (a hip joint prosthesis) was measured by both AFM and interference microscopy. Similarities in the images were seen; however, the calculated roughness was significantly different (R_q=3 and 1.5 nm). Applying a low-pass filter with a cut-off wavelength of λ_c=1.5 μm, the appearance of the images and the calculated roughness become almost identical. This strongly suggests that the two methods are consistent, and that the observed differences in shape and roughness in the nanometer range can be explained by the limited lateral resolution of the interference microscope.     

现代显微成像技术综述

概述了光学宽视场显微镜、共聚焦显微镜、超分辨率显微镜中所应用的现代显微成像技术,对各种传统和先进的显微成像原理进行了总结。光学宽视场显微镜最常用的显微技术有明场成像、暗场成像、相衬成像、偏光成像、微分干涉(DIC)成像、调制对比成像和荧光成像。相衬成像中根据不同的成像结构还有切趾相衬成像。微分干涉除了传统的偏振光照明还有圆偏振光照明(C-DIC)和专用于塑料的微分干涉(PlasDIC)。共聚焦显微镜随着计算机技术和制造技术的发展而有了巨大的发展。除了传统的共聚焦荧光显微镜以外,还有连续反斯托克斯拉曼散射(CARS)共聚焦、多光子共聚焦和白光共聚焦。超分辨率显微镜中主要介绍了受激辐射淬灭(STED)技术和紧随基态淬灭显微技术的单分子返回(GSDIM)技术。     

点扫描激光共焦显微镜物镜参数的确定

在设计点扫描激光共焦显微镜测量系统时 ,物镜参数的选择是至关重要的 ,因为它的数值孔径大小直接影响着系统的测量精度。本文对系统测量精度达到 1μm时物镜参数的选择进行了详细的阐述。     

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。     

基于数字微镜的共焦显微系统的光路设计

详细叙述了共焦技术中的横向扫描技术,介绍了基于数字微镜(DMD)的共焦显微镜结构与原理,建立了基于DMD的并行检测系统,并且进行了光路的优化设计.实验结果表明,采用传统共焦显微镜光路时,光线出射分光棱镜时存在棱镜内表面反射问题,导致DMD上同一像素块在CCD上成两个像.在此分析了上述现象产生的原理,给出了解决此问题的方...     

用于细胞三维结构观察的现代显微影像仪器及相关技术

细胞三维结构的观察分析能提供更多细胞显微水平结构和功能的信息。现代显微影像仪器及相关的三维重建等技术是研究细胞三维结构的有力工具。本文概述多种可用于细胞三维结构观察的显微工具,简单介绍其原理、优势、最新技术动态及应用领域等。     

基于MAP的自适应图像配准及超分辨率重建

图像超分辨率重建是一种将多幅低分辨率图像合成为高分辨率图像的技术.当前的超分辨重建技术主要分为图像配准和超分辨率重建2个步骤,提出一种基于最大后验概率的图像超分辨率重建算法,将这2个步骤合二为一;与此同时,为了解决配准参数以及点扩展函数估计值的不精确性问题,在每一幅低分辨率图像代价函数的残差项引入了自适应加权系数并随之...