Retrospective shading correction based on entropy minimization

Shading is a prominent phenomenon in microscopy, manifesting itself via spurious intensity variations not present in the original scene. The elimination of shading effects is frequently necessary for subsequent image processing tasks, especially if quantitative analysis is the final goal. While most of the shading effects may be minimized by setting up the image acquisition conditions carefully and capturing additional calibration images, object-dependent shading calls for retrospective correction. In this paper a novel method for retrospective shading correction is proposed. Firstly, the image formation process and the corresponding shading effects are described by a linear image formation model, which consists of an additive and a multiplicative parametric component. Secondly, shading correction is performed by the inverse of the image formation model, whose shading components are estimated retrospectively by minimizing the entropy of the acquired images. A number of tests, performed on artificial and real microscopical images, show that this approach is efficient for a variety of differently structured images and as such may have applications in and beyond the field of microscopical imaging.     

Fluorescence photobleaching-based shading correction for fluorescence microscopy

A thin fluorescent test layer, which is used in a practically mono-exponential bleaching regime, is employed to determine separately the excitation intensity and the fluorescence detection efficiency distributions in the field of view of a confocal fluorescence microscope. We demonstrate that once these distributions are known, it is possible to correct an image of a specimen for intensity variations which are caused by spatial nonuniformities of the illumination and the detection efficiency of the microscope. It is indicated that, provided a photophysically well-characterized fluorescent test layer is available, the method is potentially capable of quantifying the fluorescence intensities in an image of a specimen in terms of the fluorescence quantum yield, the absorption cross-section and the concentration of the fluorophore in the specimen.     

Fluorescence photobleaching-based image standardization for fluorescence microscopy

A method is presented for the standardization of images acquired with fluorescence microscopy, based on the knowledge of spatial distributions proportional to the microscope's absolute excitation intensity and fluorescence detection efficiency distributions over the image field. These distributions are determined using a thin fluorescent test layer, employed under practically mono-exponential photobleaching conditions. It is demonstrated that these distributions can be used for (i) the quantitative evaluation of differences between both the excitation intensity and the fluorescence detection efficiency of different fluorescence microscopes and (ii) the standardization of images acquired with different microscopes, permitting the deduction of quantitative relationships between images obtained under different imaging conditions.     

角膜神经显微图像的自适应渐晕校正

通过拼接角膜神经图像可以减小显微图像视场小的影响。由于显微图像存在渐晕效果,拼接图像会在拼接处产生伪影,影响医生诊断。为解决拼接图像的渐晕伪影问题,提出了一种通过非线性多项式函数建模进行图像渐晕校正的方法。首先,对单张角膜神经图像建立渐晕模型,设置符合渐晕物理性质的约束条件,利用L-M优化算法对渐晕模型参数进行迭代优化。在每次迭代优化过程中,计算对数信息熵,对当前渐晕模型的校正效果进行判断,防止图像过度校正。迭代优化结束后,将渐晕模型反向补偿原图像,完成渐晕校正处理。通过对比校正前后的拼接图像,校正后图像在拼接处无明显的渐晕伪影。实验测试5组不同患者的图像,校正后图像MSE、PSNR、SSIM评估指标平均值分别达到0.004 2、72.225 1 dB、0.960 0,具有最佳的校正效果。本文算法的校正效果明显优于其他同类算法的校正效果。该方法能够有效地对角膜图像渐晕效果进行校正,无须提前设置固定的相机和环境亮度参数。校正后图像拼接效果良好,可获得更加准确、清晰、视野范围大的角膜神经拼接图像。     

Aberration correction for confocal imaging in refractive-index-mismatched media

A major limitation to the use of confocal microscopes to image thick biological tissue lies in the dramatic reduction in both signal level and resolution when focusing deep into a refractive-index-mismatched specimen. This limitation may be overcome by measuring the wavefront aberration and pre-shaping the input beam so as to cancel the effects of aberration. We consider the images of planar and point objects in brightfield, single-photon fluorescence and two-photon fluorescence imaging. In all cases, the specimens are imaged using an oil-immersion objective through various thicknesses of water. The question of finite-sized pinhole is addressed and it is found, in general, that it is sufficient to correct only the first two or three orders of spherical aberration to restore adequate image signal level and optical resolution, at imaging depths of up to 50-100 wavelengths.     

Automated microscopy system for mosaic acquisition and processing

An automatic mosaic acquisition and processing system for a multiphoton microscope is described for imaging large expanses of biological specimens at or near the resolution limit of light microscopy. In a mosaic, a larger image is created from a series of smaller images individually acquired systematically across a specimen. Mosaics allow wide‐field views of biological specimens to be acquired without sacrificing resolution, providing detailed views of biological specimens within context. The system is composed of a fast‐scanning, multiphoton, confocal microscope fitted with a motorized, high‐precision stage and custom‐developed software programs for automatic image acquisition, image normalization, image alignment and stitching. Our current capabilities allow us to acquire data sets comprised of thousands to tens of thousands of individual images per mosaic. The large number of individual images involved in creating a single mosaic necessitated software development to automate both the mosaic acquisition and processing steps. In this report, we describe the methods and challenges involved in the routine creation of very large scale mosaics from brain tissue labelled with multiple fluorescent probes.     

激光诱导荧光水质监测系统的研制

提出以Nd：YAG激光器的三倍频355nm波长激光为激发光源,以单个光电倍增管（PMT）为探测器,以水体污染监测为目的的激光诱导荧光系统的研制。采用光电倍增管高速双脉冲门控与单次脉冲触发数据采样相结合新技术,有效减少了户外环境下背景光的影响,提高了系统的测量精确度。试验结果表明,该系统能接收水体表面受激发射的荧光信号,探测到的荧光信号经信号处理后与水体受污染程度有良好的相关性。     

Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy

Computational algorithms for three-dimensional deconvolution have proven successful in reducing blurring and improving the resolution of fluorescence microscopic images. However, discrepancies between the imaging conditions and the models on which such deconvolution algorithms are based may lead to artefacts and/or distortions in the images restored by application of the algorithms. In this paper, artefacts associated with a decrease of fluorescence intensity with time or slice in three-dimensional wide-field images are demonstrated using simulated images. Loss of intensity, whether due to photobleaching or other factors, leads to artefacts in the form of bands or stripes in the restored images. An empirical method for correcting the intensity losses in wide-field images has been implemented and used to correct biological images. This method is based on fitting a decreasing function to the slice intensity curve computed by summing all pixel values in each slice. The fitted curve is then used for the calculation of correction factors for each slice.