图像直方图参量规定化方法的理论分析

针对直方图参量规定化方法,分析各参量对规定直方图形状的影响,建立了规定直方图各参量与图像处理目标之间的对应关系,并通过M atlab软件对具体图片进行实验,根据实验的结果,分析得出直方图规定化的各参量在直方图处理中的作用。     

基于牙科X光片的多种直方图均衡化图像增强对比

牙科X光片在牙科疾病的诊断治疗中越来越重要。牙齿X光片机不像光学相机可以聚焦,常受低对比度和噪声的影响,很难直接借此识别疾病特征,常常需要对图片进行增强处理,故主要探讨一些适用于电子病例的牙科X光图像增强算法。算法主要有直方图均衡化、限制对比度自适应直方图均衡化、锐化中值滤波自适应直方图均衡化和改进的对比度自适应直方图均衡化方法。通过对这些算法处理后的牙科X光片图像的均方根误差(RMSE)、图像信噪比(SNR)和改善对比度指数(CII)以及视觉效果进行比较,发现用改进的对比度自适应直方图均衡化方法处理所得图像的均方根误差、改善对比度指数最小,图像信噪比最大,图像视觉效果清晰。     

Axial super resolution topography of focal adhesion by confocal microscopy

The protein organization within focal adhesions has been studied by state‐of‐the‐art super resolution methods because of its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation, limiting their application. In this report we present a phasor‐based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that with about 4,000 photon counts collected along a z‐scan, axial localization precision close to 10 nm is achievable. We show that, with within 10 nm, the axial location of paxillin, FAK, and talin is similar at focal adhesion sites, while F‐actin shows a sharp increase in height towards the cell center. We further demonstrated the live imaging capability of this method. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials. Microsc. Res. Tech., 76:1070–1078, 2013. © 2013 Wiley Periodicals, Inc.     

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.     

Moderated histogram equalization, an automatic means of enhancing the contrast in digital light micrographs reversibly

A means for improving the contrast in the images produced from digital light micrographs is described that requires no intervention by the experimenter: zero‐order, scaling, tonally independent, moderated histogram equalization. It is based upon histogram equalization, which often results in digital light micrographs that contain regions that appear to be saturated, negatively biased or very grainy. Here a non‐decreasing monotonic function is introduced into the process, which moderates the changes in contrast that are generated. This method is highly effective for all three of the main types of contrast found in digital light micrography: bright objects viewed against a dark background, e.g. fluorescence and dark‐ground or dark‐field image data sets; bright and dark objects sets against a grey background, e.g. image data sets collected with phase or Nomarski differential interference contrast optics; and darker objects set against a light background, e.g. views of absorbing specimens. Moreover, it is demonstrated that there is a single fixed moderating function, whose actions are independent of the number of elements of image data, which works well with all types of digital light micrographs, including multimodal or multidimensional image data sets. The use of this fixed function is very robust as the appearance of the final image is not altered discernibly when it is applied repeatedly to an image data set. Consequently, moderated histogram equalization can be applied to digital light micrographs as a push‐button solution, thereby eliminating biases that those undertaking the processing might have introduced during manual processing. Finally, moderated histogram equalization yields a mapping function and so, through the use of look‐up tables, indexes or palettes, the information present in the original data file can be preserved while an image with the improved contrast is displayed on the monitor screen.     

彩色遥感图像的亮度直方图局部线性化增强

针对遥感图像能量低的实际问题,提出了一种亮度直方图局部线性化图像增强方法来提高彩色遥感图像的可视效果。首先,对RGB模型描述的彩色遥感图像进行HSI变换,以有效分离H、S和I分量;其次,对亮度I分量进行传统的直方图均衡化,得到均衡化灰度映射曲线;然后,将图像梯度作为目标函数,求出最优的线性化折点位置,对灰度低端动态范围映射曲线进行线性化处理,得到局部线性化的灰度映射曲线;最后利用新的灰度映射曲线对图像进行增强处理。Himawari-8真彩色图像增强实验结果表明,经亮度直方图局部线性化增强后,像素平均梯度由73提高到了147,较传统的RGB域直方图均衡化的123及HSI域直方图均衡化的134高;图像信息熵由5.87提高到6.63,全部优于传统的RGB域直方图均衡化和HSI域直方图均衡化。本文方法有效地改善了彩色遥感图像的可视效果,提高了图像对不同目标的辨识能力。     

A method for the blind correction of the effects of attenuation and shading in light micrographs based upon moderated histogram equalization

A means of correcting for the effects of attenuation and shading in multi-dimensional, digital, light micrographs, blindly, i.e. without the need for additional control sets of image data that record these effects, is described. The method, termed trans-elemental moderated histogram equalization (TEMHE), works with all three types of image that are collected in light microscopy: bright objects viewed against a dark background, bright and dark objects set against a grey background and darker objects set against a light background. In its most simple form TEMHE requires that the features of interest are distributed widely and evenly throughout the image data. If, however, the pattern of attenuation or shading is extracted, smoothed and the result used to correct the original set of image data, then the only restriction is that when different classes of feature are present the boundaries between them are not approximately parallel to the axes of one, or more, of the dimensions to be corrected. Moreover, when it is possible to formulate a simple model of the pattern of attenuation or shading this is no longer a constraint. The method does need to analyse a large number of elements of image data (pixels, voxels, etc.) to function correctly but it will correct shading in single frames of image data providing that they are quite large and the overall signal-to-noise ratio is relatively high.     

Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy

In images acquired by confocal laser scanning microscopy (CLSM), regions corresponding to the same concentration of fluorophores in the specimen should be mapped to the same grayscale levels. However, in practice, due to multiple distortion effects, CLSM images of even homogeneous specimen regions suffer from irregular brightness variations, e.g., darkening of image edges and lightening of the center. The effects are yet more pronounced in images of real biological specimens. A spatially varying grayscale map complicates image postprocessing, e.g., in alignment of overlapping regions of two images and in 3D reconstructions, since measures of similarity usually assume a spatially independent grayscale map. We present a fast correction method based on estimating a spatially variable illumination gain, and multiplying acquired CLSM images by the inverse of the estimated gain. The method does not require any special calibration of reference images since the gain estimate is extracted from the CLSM image being corrected itself. The proposed approach exploits two types of morphological filters: the median filter and the upper Lipschitz cover. The presented correction method, tested on images of both artificial (homogeneous fluorescent layer) and real biological specimens, namely sections of a rat embryo and a rat brain, proved to be very fast and yielded a significant visual improvement. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

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 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.     

Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin

We consider various strategies for confocal imaging of human skin which seek to reduce the effects of the specimen-induced aberrations. We calculate the spherical aberration introduced by the stratified structure of skin and show how the confocal signal is affected when attempting to image at various depths within the dermis. Using simple methods it is shown how images might be improved by compensating for the induced aberration. The methods include the use of an iris to reduce the pupil area, changing the refractive index of the immersion medium and using a lens with variable coverglass correction.     

An FFT-based method for attenuation correction in fluorescence confocal microscopy

A problem in three-dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.     

Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy

Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given.     

A statistical approach for intensity loss compensation of confocal microscopy images

In this paper, a probabilistic technique for compensation of intensity loss in confocal microscopy images is presented. For single-colour-labelled specimen, confocal microscopy images are modelled as a mixture of two Gaussian probability distribution functions, one representing the background and another corresponding to the foreground. Images are segmented into foreground and background by applying Expectation Maximization algorithm to the mixture. Final intensity compensation is carried out by scaling and shifting the original intensities with the help of parameters estimated for the foreground. Since foreground is separated to calculate the compensation parameters, the method is effective even when image structure changes from frame to frame. As intensity decay function is not used, complexity associated with estimation of the intensity decay function parameters is eliminated. In addition, images can be compensated out of order, as only information from the reference image is required for the compensation of any image. These properties make our method an ideal tool for intensity compensation of confocal microscopy images that suffer intensity loss due to absorption/scattering of light as well as photobleaching and the image can change structure from optical/temporal section-to-section due to changes in the depth of specimen or due to a live specimen. The proposed method was tested with a number of confocal microscopy image stacks and results are presented to demonstrate the effectiveness of the method.     

Automated assessment of keratocyte density in clinical confocal microscopy of the corneal stroma

Cell density in the corneal stroma is typically determined by counting the number of bright objects, presumably keratocyte nuclei, in images from clinical confocal microscopy. We present a program that identifies bright objects and counts those that most likely represent cells. Selection variables were determined from 125 normal corneas with cell densities that had been assessed manually. The program was tested on 17 corneas of patients before and at several intervals to 5 years after laser in situ keratomileusis (LASIK) surgery. In these corneas, which showed a decrease in cell density after surgery, the program identified cells as well as human observers did.     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

Effects of aberrations and specimen structure in conventional, confocal and two-photon fluorescence microscopy

Specimen-induced aberrations cause a reduction in signal levels and resolution in fluorescence microscopy. Aberrations also affect the image contrast achieved by these microscopes. We model the effects of aberrations on the fluorescence signals acquired from different specimen structures, such as point-like, linear, planar and volume structures, when imaged by conventional, confocal and two-photon microscopes. From this we derive the image contrast obtained when observing combinations of such structures. We show that the effect of aberrations on the visibility of fine features depends upon the specimen morphology and that the contrast is less significantly affected in microscopes exhibiting optical sectioning. For example, we show that point objects become indistinguishable from background fluorescence in the presence of aberrations, particularly when imaged in a conventional fluorescence microscope. This demonstrates the significant advantage of using confocal or two-photon microscopes over conventional instruments when aberrations are present.     

Multicolour analysis and local image correlation in confocal microscopy

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three-dimensional (3-D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3-D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross-talk, fluorescence photobleaching and cell movements. To identify true fluorescence co-localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co-localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.     

Image sharpness and contrast transfer in coherent confocal microscopy

Confocal microscopes provide clear, thin optical sections with little disturbance from regions of the specimen that are not in focus. In addition, they appear to provide somewhat greater lateral and axial image resolution than with non-confocal microscope optics. To address the question of resolution and contrast transfer of light microscopes, a new test slide that enables the direct measurement of the contrast transfer characteristics (CTC) of microscope optics at the highest numerical aperature has been developed. With this new test slide, the performance of a confocal scanning laser microscope operating in the confocal reflection mode and the non-confocal transmission mode was examined. The CTC curves show that the confocal instrument maintains exceptionally high contrast (up to twice that with non-confocal optics) as the dimension of the object approaches the diffraction limit of resolution; at these dimensions, image detail is lost with non-confocal microscopes owing to a progressive loss of image contrast. Furthermore, we have calculated theoretical CTC curves by modelling the confocal and non-confocal imaging modes using discrete Fourier analysis. The close agreement between the theoretical and experimental CTC curves supports the earlier prediction that the coherent confocal and the incoherent non-confocal imaging mode have the same limit of resolution (defined here as the inverse of the spatial frequency at which the contrast transfer converges to zero). The apparently greater image resolution of the coherent confocal optics is a consequence of the improved contrast transfer at spacings which are close to the resolution limit.