盲复原高斯模糊图像

经典的图像恢复算法设点扩展函数(PSF)是已知的,然而在许多情况下PSF难以确定,不得不在只知道成像系统部分信息甚至没有任何信息的情况下估计真实图像和PSF,这一过程称为图像盲复原.对于高斯模糊图像,它的PSF是很难被检测出来的,因此高斯模糊图像的盲复原一直是个棘手的问题.利用高斯点扩展函数的特性,初始估计PSF并对加...     

Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive‐index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high‐end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive‐index mismatch for high‐aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high‐angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point‐scanning confocal microscope and showed that a nearly index‐matched, micron‐sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.     

Restoration of confocal images for quantitative image analysis

Quantitative studies of three-dimensional (3-D) structure of microscopic objects have been made possible through the introduction of microscopic volume imaging techniques, most notably the confocal fluorescence microscope (CFM). Although the CFM is a true volume imager, its specific imaging properties give rise to distortions in the images and hamper subsequent quantitative analysis. Therefore, it is a prerequisite that confocal images are restored prior to analysis. The distortions can be divided into several categories: attenuation of areas in the image due to self-absorption, bleaching effects, geometrical effects and distortions due to diffraction effects. Of these, absorption and diffraction effects are the most important. This paper describes a method aimed at the correction of diffraction-induced distortions. All the steps necessary in restoring confocal images are discussed, including a novel method to measure instrumental properties on a routine basis. To test the restoration procedure an image of a fluorescent planar object was restored. The results show a considerable improvement in the z-resolution and no ringing artefacts. The relevance of the method for image analysis is demonstrated by a comparison of results of applying 3-D texture analysis to restored and unrestored images of a synthetic object. Furthermore, the method can be successfully applied to noisy fluorescence images of biological objects, such as interphase cell nucei.     

Image adaptive point-spread function estimation and deconvolution for in vivo confocal microscopy

Visualizing deep inside the tissue of a thick biological sample often poses severe constraints on image conditions. Standard restoration techniques (denoising and deconvolution) can then be very useful, allowing one to increase the signal-to-noise ratio and the resolution of the images. In this paper, we consider the problem of obtaining a good determination of the point-spread function (PSF) of a confocal microscope, a prerequisite for applying deconvolution to three-dimensional image stacks acquired with this system. Because of scattering and optical distortion induced by the sample, the PSF has to be acquired anew for each experiment. To tackle this problem, we used a screening approach to estimate the PSF adaptively and automatically from the images. Small PSF-like structures were detected in the images, and a theoretical PSF model reshaped to match the geometric characteristics of these structures. We used numerical experiments to quantify the sensitivity of our detection method, and we demonstrated its usefulness by deconvolving images of the hearing organ acquired in vitro and in vivo.     

Noise suppression of point spread functions and its influence on deconvolution of three-dimensional fluorescence microscopy image sets

The point spread function (PSF) is of central importance in the image restoration of three-dimensional image sets acquired by an epifluorescent microscope. Even though it is well known that an experimental PSF is typically more accurate than a theoretical one, the noise content of the experimental PSF is often an obstacle to its use in deconvolution algorithms. In this paper we apply a recently introduced noise suppression method to achieve an effective noise reduction in experimental PSFs. We show with both simulated and experimental three-dimensional image sets that a PSF that is smoothed with this method leads to a significant improvement in the performance of deconvolution algorithms, such as the regularized least-squares algorithm and the accelerated Richardson–Lucy algorithm.     

改进的奇异值分解法估计图像点扩散函数

为了提高图像复原算法的性能,提出了一种改进的奇异值分解法估计图像的点扩散函数。从图像的退化离散模型出发,对图像进行逐层分块奇异值分解,并自动选取奇异值重组阶数以减少噪声对估计的影响。利用理想图像奇异值向量平均能谱指数模型,估计点扩散函数奇异值向量的频谱,再反傅里叶变换得到其时域结果。实验结果表明,该方法能在不同信噪比情况下估计成像系统的点扩散函数,估计结果比原有估计方法有所提高,有望为图像复原算法的预处理提供一种有效的手段。     

基于边缘预测和稀疏约束的湍流图像盲复原

大气湍流严重影响天文观测图像的成像效果,必须对退化图像进行处理才能获得清晰的图像。经典的湍流退化图像盲复原算法(IBD、NAS-RIF等)使用的先验知识过于简单,导致很多场合不能获得较优的复原效果。近几年提出的稀疏表达理论,使用自然图像边缘的稀疏先验信息指导图像复原,能复原出较多的细节,但它直接使用模糊图像的梯度图像指导点扩散函数复原,而模糊的梯度图像包含很多噪声和伪边缘,无效的梯度会误导点扩散函数的估计,从而使复原图像中出现较多伪迹。针对上述问题,提出了一种基于边缘预测和稀疏比值正则约束的湍流退化图像盲复原算法,该算法首先从当前的复原图像中预测出有效的边缘,然后将边缘预测信息与自然图像边缘的稀疏先验信息相结合指导点扩散函数复原,得到点扩散函数后,再通过一种非盲复原算法恢复出当前的目标图像,并将此复原图像作为下一次边缘预测的输入图像,如此迭代循环直到求出最终清晰的目标图像。所提算法结合了图像的先验信息与退化图像自身包含的有效信息,能有效抑制图像复原过程中产生的伪迹,获得令人满意的结果。针对多幅模拟的湍流退化图像进行仿真测试,验证了算法的有效性。     

Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements

A modified fluorescence correlation microscope (FCM) was built on a commercial confocal laser scanning microscope (CLSM) by adding two sensitive detectors to perform fluorescence correlation spectroscopy (FCS). A single pinhole for both imaging and spectroscopy and a simple slider switch between the two modes thus facilitate the accurate positioning of the FCS observation volume after the confocal image acquisition. Due to the use of a single pinhole for CLSM and FCS the identity of imaged and spectroscopically observed positions is guaranteed. The presented FCM system has the capability to position the FCS observation volume at any point within the inner 30% of the field of view without loss in performance and in the inner 60% of the field of view with changes of FCS parameters of less than 10%. A single pinhole scheme for spatial fluorescence cross correlation spectroscopy performed on the FCM system is proposed to determine microfluidic flow angles. To show the applicability and versatility of the system, we measured the translational diffusion coefficients on the upper and lower membranes of Chinese hamster ovary cells. Two-photon excitation FCS was also realized by coupling a pulsed Ti: sapphire laser into the microscope and used for flow direction characterization in microchannels.     

Simultaneous degradation estimation and restoration of confocal images and performance evaluation by colocalization analysis

A novel method for joint restoration and estimation of the degradation of confocal microscope images is presented. The observed images are degraded due to two sources: blurring due to the band-limited nature of the optical system [modelled by the point spread function (PSF)], and Poisson noise contaminates the observations due to the discrete nature of the photon detection process. The proposed method iterates noise reduction, blur estimation and deblurring, and applies these steps in two phases, i.e. a training phase and a restoration phase. In the first phase, these three steps are iterated until the blur estimation converges. Noise reduction and blur estimation are performed using steerable pyramids, and the deblurring is performed by the Richardson–Lucy algorithm. The second phase is the actual restoration. From then on, the blur estimation is used as a criterion to measure the image quality. The iterations are stopped when this measure converges, a result that is guaranteed. The integrated method is completely automatic, and no prior information on the image is required. The method has been given the name SPERRIL (Steerable Pyramid-based Estimation and Regularized Richardson–Lucy restoration). Compared with existing techniques by both objective measures and visual observation, in the SPERRIL-restored images noise is better suppressed.     

Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration

A set of methods leading to volume reconstruction of biological specimens larger than the field of view of a confocal laser scanning microscope (CLSM) is presented. Large tissue specimens are cut into thin physical slices and volume data sets are captured from all studied physical slices by CLSM. Overlapping spatial tiles of the same physical slice are stitched in horizontal direction. Image volumes of successive physical slices are linked in axial direction by applying an elastic registration algorithm to compensate for deformations because of cutting the specimen. We present a method enabling us to keep true object morphology using a priori information about the shape and size of the specimen, available from images of the cutting planes captured by a USB light microscope immediately before cutting the specimen by a microtome. The errors introduced by elastic registration are evaluated using a stereological point counting method and the Procrustes distance. Finally, the images are enhanced to compensate for the effect of the light attenuation with depth and visualized by a hardware accelerated volume rendering. Algorithmic steps of the reconstruction, namely elastic registration, object morphology preservation, image enhancement, and volume visualization, are implemented in a new Rapid3D software package. Because confocal microscopes get more and more frequently used in scientific laboratories, the described volume reconstruction may become an easy‐to‐apply tool to study large biological objects, tissues, and organs in histology, embryology, evolution biology, and developmental biology. In this work, we demonstrate the reconstruction using a postcranial part of a 17‐day‐old laboratory Wistar rat embryo. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

Absorption and scattering correction in fluorescence confocal microscopy

In three-dimensional (3-D) fluorescence images produced by a confocal scanning laser microscope (CSLM), the contribution of the deeper layers is attenuated due to absorption and scattering of both the excitation and the fluorescence light. Because of these effects a quantitative analysis of the images is not always possible without restoration. Both scattering and absorption are governed by an exponential decay law. Using only one (space-dependent) extinction coefficient, the total attenuation process can be described. Given the extinction coefficient we calculate within a non-uniform object the relative intensity of the excitation light at its deeper layers. We also give a method to estimate the extinction coefficients which are required to restore 3-D images. An implementation of such a restoration filter is discussed and an example of a successful restoration is given.     

Isotropic high-resolution three-dimensional confocal micro-rotation imaging for non-adherent living cells

Recently, micro-rotation confocal microscopy has enabled the acquisition of a sequence of micro-rotated images of nonadherent living cells obtained during a partially controlled rotation movement of the cell through the focal plane. Although we are now able to estimate the three-dimensional position of every optical section with respect to the cell frame, the reconstruction of the cell from the positioned micro-rotated images remains a last task that this paper addresses. This is not strictly an interpolation problem since a micro-rotated image is a convoluted two-dimensional map of a three-dimensional reality. It is rather a 'reconstruction from projection' problem where the term projection is associated to the PSF of the deconvolution process. Micro-rotation microscopy has a specific difficulty. It does not yield a complete coverage of the volume. In this paper, experiments illustrate the ability of the classical EM algorithm to deconvolve efficiently cell volume despite of the incomplete coverage. This cell reconstruction method is compared to a kernel-based method of interpolation, which does not take account explicitly the point-spread-function (PSF). It is also compared to the standard volume obtained from a conventional z-stack. Our results suggest that deconvolution of micro-rotation image series opens some exciting new avenues for further analysis, ultimately laying the way towards establishing an enhanced resolution 3D light microscopy.     

湍流退化红外图像降晰函数辨识

针对高速湍流场导致的红外成像模糊,提出了一种基于图像质量评价的湍流退化红外图像降晰函数辨识算法。利用先验知识将退化过程简化为可用参数描述的二维高斯函数形式,将退化图像分割为边缘区、纹理区和平坦区,计算不同支持域下复原图像在不同参数时的峰度值;利用曲率最大准则对得到的“峰度-参数”曲线进行相应的降晰函数参数估计,进而由支持域和对应的估计参数得到对应降晰函数并用于复原退化图像;最后对复原图像进行无参考图像质量评价,评价指标最高的复原图像对应的降晰函数即为最终辨识结果。实验结果表明：该算法能较好地辨识降晰函数参数和支持域大小,当退化图像信噪比大于30 dB时,估计参数与真实值的最大偏差小于±5%。该算法所得结果可以作为湍流退化红外图像其他复原算法的降晰函数起始估计。     

A confocal laser microscope scanner for digital recording of optical serial sections

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illumination, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinates scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024 × 1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (～ 1 μm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.     

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.     

Single-pinhole confocal imaging of sub-resolution sparse objects using experimental point spread function and image restoration

Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three-dimensional imaging of subresolution sparse objects. The experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 nm. This amounts to 1. 43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used.     

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.     

改善激光共焦扫描显微镜像质的方法

为解决传统光学显微镜样本上每一点的图像都受到邻近点衍射或散射光干扰的问题,研发了一套基于C#WinForm控制平台进行连续扫描方式的激光共焦扫描显微镜(LCSM)系统,并且成功地对生物细胞进行了扫描成像。针对共焦显微镜图像像质不高的问题,提出合理选取探测器针孔直径,并通过高斯低通滤波、盲解卷积的方法,确保实现高像质。实验结果表明,基于上述方法改进后的LCSM具有较高图像质量,该方法简单易行,便于实施。     

PHOEBE,a prototype scanning laser-feedback microscope for imaging biological cells in aqueous media

Based on the principle of laser-feedback interferometry (LFI), a laser-feedback microscope (LFM) has been constructed capable of providing an axial (z) resolution of a target surface topography of ～ 1 nm and a lateral (x, y) resolution of ～ 200 nm when used with a high-numerical-aperture oil-immersion microscope objective. LFI is a form of interferometry in which a laser's intensity is modulated by light re-entering the illuminating laser. Interfering with the light circulating in the laser resonant cavity, this back-reflected light gives information about an object's position and reflectivity. Using a 1-mW He–Ne (λ = 632·8 nm) laser, this microscope (PHOEBE) is capable of obtaining 256 × 256-pixel images over fields from (10 μm × 10 μm) to (120 μm × 120 μm) in ～ 30 s. An electromechanical feedback circuit holds the optical pathlength between the laser output mirror and a point on the scanned object constant; this allows two types of images (surface topography and surface reflectivity) to be obtained simultaneously. For biological cells, imaging can be accomplished using back-reflected light originating from small refractive-index changes (> 0·02) at cell membrane/water interfaces; alternatively, the optical pathlength through the cell interior can be measured point-by-point by growing or placing a cell suspension on a higher-reflecting substrate (glass or a silicon wafer). Advantages of the laser-feedback microscope in comparison to other confocal optical microscopes include: the simplicity of the single-axis interferometric design; the confocal property of the laser-feedback microscope (a virtual pinhole), which is achieved by the requirement that only light that re-enters the laser meeting the stringent frequency, spatial (TEM₀₀), and coherence requirements of the laser cavity resonator mode modulate the laser intensity; and the improved axial resolution, which is based on interferometric measurement of optical amplitude and phase rather than by use of a pinhole as in other types of confocal microscopes.     

点衍射波前位相的测评

提出一种检测点衍射干涉仪关键部件针孔所产生的衍射光学波前的方法。介绍了点衍射波前的产生原理,分析了小孔质量状态、照明光路调整状态与波前各个像差分量之间的关系。基于信息光学基础理论,采用傅里叶变换和迭代算法,采集针孔衍射图像并进行计算分析,实现对衍射波前的位相复原以获得波前信息。阐述了测试方法的理论依据和计算公式,应用研制的位相复原分析计算软件测试并分析了实际采集的点衍射图像,通过15次的迭代,输出的位相值逐渐收敛,图像误差因子下降到0.12。目前,该方法已用于对针孔的筛选和针孔照明系统的装调中,实验结果证明了该测试方法的可行性。