Measurement and restoration of the point spread function of fluorescence confocal microscopy

The point spread function of an objective lens of a fluorescence confocal microscope was directly measured by imaging fluorescent beads. We analysed how the measurement of the point spread function was influenced by the diameter of the fluorescent beads and how the restoration technique with a deconvolution algorithm improved the measuring performance. Numerical and experimental results are presented for a typical point spread function and a zero‐centred point spread function.     

Three-dimensional point spread function model for line-scanning confocal microscope with high-aperture objective

Point Spread Function (PSF) modelling is an important task in image formation analysis. In confocal microscopy, the exact PSF is rarely known, thus one has to rely on its approximation. An initial estimation is usually performed experimentally by measuring fluorescent beads or analytically by studying properties of the optical system. Yet, fluorescent line‐scanning confocal microscopes are not widespread; therefore, very few adapted models are available in the literature. In this paper, we propose an analytical PSF model for line‐scanning confocal microscopes. Validation is performed by measuring the error between our model and an experimental PSF measured with fluorescent beads, assumed to represent the real PSF. Comparison with existing models is also presented.     

Three-dimensional image restoration and super-resolution in fluorescence confocal microscopy

Confocal scanning laser microscopy (CSLM) provides optical sectioning of a fluorescent sample and improved resolution with respect to conventional optical microscopy. As a result, three-dimensional (3-D) imaging of biological objects becomes possible. A difficulty is that the lateral resolution is better than the axial resolution and, thus, the microscope provides orientation-dependent images. However, a theoretical investigation of the process of image formation in CSLM shows that it must be possible to improve the resolution obtained in practice. We present two methods for achieving such a result in the case of 3-D fluorescent objects. The first method applies to conventional CSLM, where the image is detected only on the optical axis for any scanning position. Since the resulting 3-D image is the convolution of the object with the impulse-response function of the instrument, the problem of image restoration is a deconvolution problem and is affected by numerical instability. A short introduction to the linear methods developed for obtaining stable solutions of these problems (the so-called regularization theory of ill-posed problems) is given and an application to a real image is discussed. The second method applies to a new version of CSLM proposed in recent years. In such a case the full image must be measured by a suitable array of detectors. For each scanning position the data are not single numbers but vectors. Then, in order to recover the object, one must solve a Fredholm integral equation of the first kind. A method for the solution of this equation is presented and the possibility of achieving super-resolution is demonstrated. More precisely, we show that it is possible to improve by about a factor of 2 the resolution of conventional CSLM both in the lateral and axial directions.     

Imaging properties of bright-field and annular-dark-field scanning confocal electron microscopy: II. point spread function analysis

The imaging properties of bright field and annular dark field scanning confocal electron microscopy (BF-SCEM and ADF-SCEM) are discussed based on their point spread functions (PSFs) in comparison with multislice simulations. Although the PSFs of BF-SCEM and ADF-SCEM show similar hourglass shapes, their numerical distributions are quite different: BF-SCEM PSF is always positive and shows a center of symmetry whereas the ADF-SCEM PSF is complex and has Hermitian symmetry. These PSF properties explain the large elongation effect in BF-SCEM for laterally extended object and almost no-elongation in ADF-SCEM, illustrating the importance of the numerical analysis of PSFs. The Hermitian symmetry of the ADF-SCEM PSF results in an interesting “edge enhancement effect” at the interface. Simulation using the PSF and the multislice method verified this effect at GaAs surfaces and InAs interfaces embedded in GaAs. This unique feature of ADF-SCEM can potentially be useful for depth sectioning. It is also pointed out that a PSF imaging model cannot be applicable for BF-SCEM of a phase object, when the system is symmetric and aberration free.     

基于稀疏表示和Weber定律的运动图像盲复原

针对运动过程中视觉图像易产生运动模糊的问题,提出了一种基于稀疏表示和Weber定律相结合的图像盲复原方法。该方法利用冲击滤波器预测模糊图像的显著边缘梯度,并用多尺度策略由粗到细进行模糊核的估计。然后,对图像盲复原模型进行稀疏正则化约束,并结合反映人类视觉特性的Weber定律对合成模糊图像和真实模糊图像进行盲复原。实验结果表明,本文采用的盲复原算法的性能指标和图像的纹理都达到了较优的复原效果。与近年较好的Rob Fergus去模糊方法和Xu Li去模糊方法相比,对Lena模糊图去模糊后的结构相似度(SSIM)为0.762 4,峰值信噪比(PSNR)提高了1.82~2.99dB;对Cameraman模糊图去模糊后的结构相似度(SSIM)为0.8589,PSNR提高了2.46~5.58dB。另外,本文方法降低了复原图像的边界伪影,符合人的视觉感知特性。     

用于生物组织层析成像的共聚焦内窥镜

为了获取生物组织的层析图像,建立了共聚焦内窥镜成像系统,对该系统的横向和轴向分辨能力进行测量和分析,并且对其在猪皮肤组织的层析成像能力进行研究。首先根据系统工作要求,说明系统的工作原理和组成结构,然后给出系统空间分辨能力的理论计算式,最后测试系统性能,说明测量值与理论值存在偏差的主要原因。实验结果表明：建立的系统轴向分辨能力约为10 ;横向分辨能力约为1.9 ;该系统可以对猪皮肤组织进行层析成像。     

组合曝光的计算成像系统及其复原

传统光学成像方式在低照度条件下拍摄高速运动物体时,通过积分时间的配置难以平衡能量获取和高速运动模糊之间的矛盾.为了解决低照度条件下高速运动物体的清晰成像问题,本文提出一种基于组合曝光图像的计算成像方式,通过高帧频相机采集邻近两帧形成组合曝光图像对,基于两帧图像信息的互补,合理估算运动模糊点扩散函数,最终获取高信噪比的复...     

A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.     

基于L0稀疏先验的相机抖动模糊图像盲复原

提出了一种基于L0稀疏先验的改进正则化模糊图像盲复原算法来解决相机抖动所产生的模糊问题。根据模糊图像的梯度分布要比清晰图像稠密并且暗通道的稀疏性也相对较小这一固有属性建立了新的优化模型。针对L0范数的高度非凸性和暗通道稀疏优化过程中涉及到的非线性最小化问题,提出了一种近似线性映射矩阵,并用半二次分解法对L0最小化问题进行求解。最后,采用快速傅里叶变换在频域中对模糊核及清晰图像进行交替迭代运算得到复原图像。对多幅不同类型的模糊图像进行了实验,结果显示:复原图像平均灰度梯度高达11.411,图像信息熵达到7.304,处理365×285的图像只需8.07s。提出的算法有效抑制了图像边缘处的振铃效应,完整保留了清晰的细节信息的同时显著提高了运算速度,并适用于多种不同类型图像的盲复原。     

含噪声模糊图像的点扩展函数参数辨识

针对运动模糊和散焦模糊,提出了一种基于双谱的对含有噪声的模糊图像点扩展函数参数辨识的方法。首先计算出一幅标准测试图像经模糊后的双谱,之后通过曲线拟合出双谱中的统计特性与模糊尺度之间的函数关系,由此训练出的BP神经网络可以完成对其它含有噪声的模糊图像的点扩展函数的参数辨识。实验结果表明,本方法适用于含有噪声的一定模糊参数范围内的散焦模糊和运动模糊图像,在信噪比为25dB的情况下,辨别出模糊尺度的偏差不超过0.5个像素。     

A wavefront generator for complex pupil function synthesis and point spread function engineering

We describe a simple method to produce an arbitrary complex optical field using a ferroelectric liquid crystal spatial light modulator. The system is configured so as to act as a pupil plane filter in a confocal microscope. The ability to tune the complex pupil function permits the system to be used both to modify the imaging performance by effectively engineering the point spread function as well as to remove optical aberrations present in the optical system.     

Three dimensional image restoration in fluorescence lifetime imaging microscopy

A microscope set-up and numerical methods are described which enable the measurement and reconstruction of three-dimensional nanosecond fluorescence lifetime images in every voxel. The frequency domain fluorescence lifetime imaging microscope (FLIM) utilizes phase detection of high-frequency modulated light by homodyne mixing on a microchannel plate image intensifier. The output signal at the image intensifier's phosphor screen is integrated on a charge coupled device camera. A scanning stage is employed to obtain a series of phase-dependent intensity images at equally separated depths in a specimen. The Fourier transform of phase-dependent data gives three-dimensional (3D) images of the Fourier coefficients. These images are deblurred using an Iterative Constrained Tikhonov–Miller (ICTM) algorithm in conjunction with a measured point spread function. The 3D reconstruction of fluorescence lifetimes are calculated from the deblurred images of the Fourier coefficients. An improved spatial and temporal resolution of fluorescence lifetimes was obtained using this approach to the reconstruction of simulated 3D FLIM data. The technique was applied to restore 3D FLIM data of a live cell specimen expressing two green fluorescent protein fusion constructs having distinct fluorescence lifetimes which localized to separate cellular compartments.     

Automated imaging of extended tissue volumes using confocal microscopy

Confocal microscopy enables constitutive elements of cells and tissues to be viewed at high resolution and reconstructed in three dimensions, but is constrained by the limited extent of the volumes that can be imaged. We have developed an automated technique that enables serial confocal images to be acquired over large tissue areas and volumes. The computer-controlled system, which integrates a confocal microscope and an ultramill using a high-precision translation stage, inherently preserves specimen registration, and the user control interface enables flexible specification of imaging protocols over a wide range of scales and resolutions. With this system it is possible to reconstruct specified morphological features in three dimensions and locate them accurately throughout a tissue sample. We have successfully imaged various samples at 1-mum voxel resolution on volumes up to 4 mm3 and on areas up to 75 mm2. Used in conjunction with appropriate embedding media and immuno-histochemical probes, the techniques described in this paper make it possible to routinely map the distributions of key intracellular structures over much larger tissue domains than has been easily achievable in the past.     

The three-dimensional point spread functions of a microscope objective in image and object space

The three-dimensional point spread function (3-D PSF) of an optical system in image space is distinguished from the 3-D PSF in object space and the relation between the two 3-D PSFs is derived. By using this relation one 3-D PSF can be easily obtained from the other. The 3-D PSFs are given in a single integral expression, which can be computed numerically. The results of this study can be used in 3-D image processing for microscopy and have been applied to the analysis of the diffusion of fluorescent molecules in a 3-D porous medium.     

结合引导滤波和卷积稀疏表示的红外与可见光图像融合

为了解决红外与可见光图像融合时信息容易相互干扰、影响融合质量的问题,将引导滤波、高斯低通滤波与非下采样方向滤波器组相结合,提出一种新的图像融合方法。利用引导滤波和高斯低通滤波,将源图像分解为低频近似部分、强边缘部分和高频细节部分,并将高频细节部分进行非下采样方向滤波,进一步得到高频方向细节部分;对低频近似部分应用基于局部区域能量的融合规则,对强边缘部分提出一种基于卷积稀疏表示的融合规则,对高频方向细节部分提出改进的脉冲耦合神经网络的融合规则,得到相应的融合部分,并通过逆变换得到最终的融合图像。对多组红外与可见光图像的实验结果表明,算法得到的融合结果的主观视觉效果和客观评价指标均优于传统的图像融合方法,其客观评价指标中的标准差、信息熵、互信息、平均梯度和空间频率相比融合效果较好的基于离散小波变换和稀疏表示的融合方法平均提高20.28%、2.24%、47.41%、5.34%、8.02%。     

Three-dimensional imaging of corneal cells using in vivo confocal microscopy

Confocal microscopy is a unique and powerful imaging paradigm which allows optical sectioning through intact tissue. Real-time tandem scanning confocal microscopy has previously been used to generate high-magnification two-dimensional (2-D) images of cells in living organ systems. Inherent problems with movement, however, have prevented the in vivo acquisition of complete 3-D datasets. The development of a new objective lens, used in combination with specialized real-time image acquisition procedures, has allowed sequential serial sections to be obtained in vivo from the rabbit cornea for the first time. These sections can be digitially registered and stacked on the computer to provide a 3-D reconstruction of the corneal cells. This technique should serve as a useful method for studying 3-D structures and analysing 4-D phenomena at the cellular level in living animals. Three-dimensional images of a stromal nerve in normal rabbit cornea and of fibroblasts within a rabbit corneal wound are presented as examples of current capabilities.     

In vivo imaging of human teeth and skin using real-time confocal microscopy

Confocal microscopy is currently being used to obtain images with higher lateral and axial resolution than conventional light microscopic techniques. Most current confocal microscopic applications describe the use of in vitro preparations. The tandem scanning microscope (TSM) can be applied in the in vivo microscopic evaluation of living tissue. This article discusses in vivo applications of the TSM in the study of human teeth and skin.     

四波段共光轴成像实验平台及其图像融合

为获取以及充分利用场景的多波段信息,同时为多波段图像算法研究奠定基础,基于非制冷红外焦平面阵列,研制了共光轴分束的可见光+近红外、短波红外、中波红外和长波红外四波段光电成像实验平台.给出了基于四波段光电成像实验平台的图像处理案例,使用现场可编程门阵列完成各路图像的预处理与多波段图像融合.预处理过程主要包括可见光机芯盲元...