GELFISH – Graphical environment for labelling fluorescence in-situ hybridization images

Signal (dot) counting in fluorescence in-situ hybridization (FISH) images that relies on an automatic focusing method for obtaining clearly defined images is a time-consuming procedure prone to errors. Our recently developed system has dispensed with automatic focusing, and instead relies on a neural network classifying focused and unfocused signals into valid and artefact data, respectively, and thereby discriminating between in- and out-of-focus images. However, to train the classifier accurate labelling of the image signals is required. GELFISH is a Graphical Environment for Labelling FISH images that enables the rejection of unanalysable nuclei and labelling of FISH signals simply and rapidly. GELFISH is flexible and can be modified easily for additional FISH applications. Also, implemented using popular software, the environment can be employed on any computer by any user. Finally, GELFISH is proposed in controlling a classifier-based dot counting system.     

Analog enhancement of videomicroscope images

A simple, inexpensive technique for enhancing the contrast and resolution of videomicroscope images has been developed. The system has manual controls for gain and pedestal (black level) which permit expansion of low contrast images to the full white-to-black video range. Analog delay-line based circuits are used to sharpen the edges and enhance fine details in the image. These circuits also produce an effective increase in the information content of the image by selectively amplifying low amplitude, high frequency components of the video signal. When live, unstained cells were examined at high magnifications, cytoplasmic structures which were only faintly visible in the unenhanced image became clear. The images of fluorescent objects appear in pseudo-relief, which improves visibility even in the presence of background fluorescence. The system enhances images by performing signal processing functions that otherwise require expensive digital image processing equipment.     

基于荧光原位杂交技术的全自动病理染色系统结构设计

荧光原位杂交(FISH)技术是检测恶性肿瘤细胞核酸序列的常用方法,在癌症的诊疗领域有广泛应用。为实现FISH实验的全自动化,设计了一种全自动病理染色系统。该系统设置有多轴操纵臂、试剂加样器、载玻片、盖玻片夹具、辅助模块等。多轴操纵臂定位误差不大于±0.1 mm,提取及丢弃移液枪头准确率大于99.5%,抽取盖玻片成功率大于99%,微量试剂加样误差不大于0.6μL,大量试剂加样误差不大于0.5 mL,载玻片夹具控温误差不大于±1℃且无试剂泄漏。生物实验结果表明,该系统荧光染色效果良好,信号点清晰可见,荧光图像可判读率超过90%,具有较好的人工替代性。     

Cytogenetic profiling by FISH microscopy and comparison with light microscopy and complete blood count findings in myelodysplastic syndrome

Karyotype, bone marrow blast percentage and cytopenia influence the prognosis of myelodysplastic syndrome. We studied the abnormalities detected by fluorescence in situ hybridization (FISH) in myelodysplastic syndrome and associated haematological profile with abnormalities detected by FISH. Complete blood counts, peripheral blood and bone marrow of patients were evaluated for cytopenia, dysplasia and blasts. FISH probes were used to detect del(5q), gain of chromosome 8, de (7q/‐7) and del(20 q). Multiple regression analysis was used to study the association of FISH abnormalities, age and sex with haematological profile. Mc Nemar's test studied the relationship between FISH abnormalities and dysplastic features in bone marrow. Cytogenetic abnormalities were detected by FISH in 25.7% of patients. Del(20 q) was seen in 14.2% of patients. FISH was able to predict changes in peripheral blood blast count by 80% (p ? 0.0001). Cytogenetic abnormalities were not seen in 74.2% of patients. Groups with FISH abnormalities have a different haematological profile, and these abnormalities have a significant effect on blast percentage.     

Four-dimensional factor analysis of confocal image sequences (4D-FAMIS) to detect and characterize low copy numbers of human papillomavirus DNA by FISH in HeLa and SiHa cells

Visualization and localization of specific DNA sequences were performed by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), and four-dimensional factor analysis of biomedical image sequences (4D-FAMIS). HeLa and SiHa cells containing, respectively 20–50 and 1–2 copies per cell of human papillomavirus (HPV) DNA type 18 and 16 integrated in cellular DNA were used as models. HPV-DNA was identified using DNA probes containing the whole genome of HPV-DNA type 18 or 16, and DNA–DNA hybrids were revealed by alkaline phosphatase and Fast Red. Cell nuclei were counterstained with thiazole orange (TO) or TOTO-iodide. 4D image sequences were obtained using successive dynamic or spectral sequences of images on different optical sections from CLSM. The location of fluorescent signals within the preparations was determined by FAMIS. This original method summarizes image sequences into a reduced number of images called factor images, and curves called factors. Factors estimate different individual physical behaviours in the sequence such as extinction velocity, spectral patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. We distinguished between Fast Red and nucleus stainings in HPV-DNA hybridization signals by taking into account differences in their extinction velocities (fluorescence decay rate) or spectral patterns, and in their focus (depth emission profiles). In HeLa cells, factor images showed that Fast-Red-stained targets could be distinguished from nucleus stainings, and were located on different focal planes of the nuclei. In SiHa cells, 4D-FAMIS determined as few as 1–2 copies per cell of HPV-DNA type 16 located in continuous focal planes. Therefore, 4D-FAMIS, together with CLSM, made the detection and characterization of low copy numbers of genes in whole cells possible.     

A simple twist for signal enhancement in non-linear optical microscopy

We describe a very simple but elegant approach to two-photon fluorescence signal enhancement by intensity modulation with immediate application in two-photon laser-scanning fluorescence microscopy. This method of enhancement shows potential application in any microscopic technique that result from non-linear photon absorption and plays a pivotal role in live cell imaging.     

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.     

基于小波分解灰关联的热波检测图像增强

针对热波检测图像存在的高噪声、低对比度等问题,提出一种基于小波分解和灰关联分析的图像增强方法。该方法首先采用小波变换对待处理的热波图像进行三级小波分解,得到图像相应的低频分量和高频分量,然后利用图像中干扰信号和有用信号在分解后不同分量上的分布规律,采用灰色理论中的灰色关联分析理论来区分高频分量中的干扰信号和有用信号,从而实现对图像中噪声的抑制以提高图像的质量。实验结果表明:提出的方法与常规的滤波方法、小波阈值去噪增强等方法相比,图像的对比度得到明显改善,峰值信噪比最大,因此该方法可用于热波检测图像的增强处理中。     

Bremsstrahlung enhancement in electron probe microanalysis for homogeneous samples using Monte Carlo simulation

Fluorescence enhancement in samples irradiated in a scanning electron microscope or an electron microprobe should be appropriately assessed in order not to distort quantitative analyses. Several models have been proposed to take into account this effect and current quantification routines are based on them, many of which have been developed under the assumption that bremsstrahlung fluorescence correction is negligible when compared to characteristic enhancement; however, no concluding arguments have been provided in order to support this assumption. As detectors are unable to discriminate primary from secondary characteristic X‐rays, Monte Carlo simulation of radiation transport becomes a determinant tool in the study of this fluorescence enhancement. In this work, bremsstrahlung fluorescence enhancement in electron probe microanalysis has been studied by using the interaction forcing routine offered by penelope 2008 as a variance reduction alternative. The developed software allowed us to show that bremsstrahlung and characteristic fluorescence corrections are in fact comparable in the studied cases. As an extra result, the interaction forcing approach appears as a most efficient method, not only in the computation of the continuum enhancement but also for the assessment of the characteristic fluorescence correction.     

胶囊内窥系统的像质提升方法

针对目前胶囊内窥镜存在分辨率低,视场角有限,受噪声影响大等问题,提出了系统解决方案。通过引入Q-type非球面校正像差,得到一款全视场角为160°,相对孔径为F#3.0,系统总长为4.3 mm的内窥镜成像镜头,在140 lp/mm处各视场调制传递函数（MTF）值均大于0.3。光学系统成像质量的好坏不仅取决于镜头的性能,还与图像传感器有关,尤其在低照度环境下。通过分析传感器工作时各个阶段噪声的特性得到噪声模型。利用建立的噪声模型,合成了胶囊内窥镜图像数据集,并训练神经网络模型。对算法模型的测试结果表明,本文提出的综合解决方案可以有效地提高胶囊内窥镜系统的成像质量。     

Image analysis for denoising full-field frequency-domain fluorescence lifetime images

Video-rate fluorescence lifetime-resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full-field frequency-domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video-rate image acquisition is possible. Homodyne FLIM uses a gain-modulated image intensified charge-coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super-resolution imaging).     

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1. 77. p1–79 and the plasmid probe α-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.     

Low‐temperature glycol methacrylate resin embedding method: A protocol suitable for bone marrow immunohistochemistry,PCR, and fish analysis

Molecular analyses such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) are demanded to improve diagnostic accuracy in addition to immunohistopathology of bone marrow (BM) trephine specimens. Conventional BM embedding method needs decalcification, and its procedure may impair tissue morphology and DNA quality. Here, we report an undecalcified method by which glycol methacrylate resin is polymerized at low temperature (4°C). Using this method, BM enzyme activity and antigenic determinants are well preserved, and moreover, DNA extracted from plastic embedding sections is suitable for PCR amplification and sequencing, FISH analysis can be well done because of the DNA integrity of BM sections. If working with BM trephine specimen, our protocol offers the possibility to combine superior morphology with modern molecular analysis. Microsc. Res. Tech. 73:1067–1071, 2010. © 2010 Wiley‐Liss, Inc.     

Image analysis of particle dispersions in microscopy images of cryo-sectioned sausages

Two feature extraction methods, the three-dimensional (3-D) local box-counting method and the area distribution method, are presented to describe the fat dispersion pattern on digital microscopy images of cryo-sectioned sausages. Both methods calculate whole arrays of variables for each microscopy image. The 3-D box-counting method calculates scale dependent (local) dimensions. This is in contrast to common fractal methods, which are univariate. Principal component analysis (PCA) was used to show that different sausages yield different fat dispersion patterns. Partial least square regression (PLS) shows that there is a correlation between the variables gained with both methods and the fat content.     

航空摆扫相机转弯成像像移分析及补偿

为保证航空摆扫相机转弯成像过程中的成像质量,对其像移计算及补偿方法进行了研究。根据航空摆扫相机的成像原理,利用几何建模及速度矢量分解建立了转弯成像像移计算模型,给出了基于均值补偿的转弯前向像移补偿方法。转弯前向像移补偿分析表明:相机焦距为500 mm,曝光时间为0.01 s,速高比为0.02 rad/s,纵向视场角为10°,转弯角速度为0.5(°)/s时,最大前向像移补偿残差量为2.22μm;转弯角速度为1.5(°)/s时,最大前向像移补偿残差量为3.36μm。另外,转弯横向像移补偿分析表明:横向像移量随纵向视场角幅值的增加而增大,曝光时间为0.005 s,横向视场角为30(°),转弯角速度为1(°)/s时,横向像移量在纵向视场角为4.5°时达到3μm。转弯成像试飞实验结果表明:得到的图像像质优良,无几何形变,前向像移补偿良好,验证了本文提出的转弯成像像移补偿方法的正确性。     

多图像融合Retinex用于弱光图像增强

为解决弱光图像增强过程中对比度增强和自然度保持问题,本文提出一种基于Retinex的多图像自适应加权最小二乘滤波算法。首先,在图像的每个像素的R,G,B三通道中找到最大亮度值作为该像素的初始照明估计,根据Retinex理论生成反射图像,并通过形态学闭合方式调整反射图;接着,在初始照明图基础上,通过Gamma变换和双对数变换方法分别生成全局对比度增强图和局部自然度保持照明图;随后,设计一种自适应加权最小二乘滤波融合策略将三幅照明图融合成最终照明估计图;最后合成上述的最终照明图和调整反射图以获得弱光增强后的图像。实验结果表明,本文所提出算法的亮度顺序差(LOE)及盲图像质量评价(NIQE)值更低,可同时降低到4.12和3.25,较其他方法表现出更好的增强效果。证明了本文算法能有效地增强弱光图像对比度,同时保持图像自然度。     

面向机柜表面缺陷检测的不均匀光照和低亮度图像增强方法

光照条件是大尺寸机柜表面缺陷检测的重要影响因素。当光照分布不均匀或光照强度不足时,采集得到的机柜表面图像质量低,造成缺陷检测误差。为此,提出一种融合卡通纹理分解和最优双曲正切曲线的图像增强方法。首先,采用导向滤波将机柜表面图像分解为卡通图和纹理图,利用高斯尺度空间理论建立光照模型,实现不均匀光照去除;其次,研究图像的双曲正切曲线性质,通过图像加权拉伸实现低亮度图像增强;最后,采用对比度、亮度和灰度方差乘积对图像增强效果进行评价,同时对增强前和增强后的图像进行缺陷检测,进行对比分析验证。实验结果表明,该方法能实现光照不均且低亮度的机柜表面图像增强,机柜表面缺陷检测的准确率显著提升,召回率提高了29%,F值提高了21%。     

马尔科夫随机场模型下的Retinex夜间彩色图像增强

由于Retinex算法在处理夜间彩色图像时容易出现光晕、颜色失真、细节丢失与噪声干扰等问题,本文基于马尔科夫随机场（MRF）提出了一种针对单幅图像的Retinex图像增强算法。该算法在HSV颜色空间下采用线性引导滤波估计图像照度分量;在MRF模型下求解仅包含物体本身特性的反射分量,并通过颜色恢复函数与增益补偿方法进行颜色恢复与校正,最终实现了夜间彩色图像的增强。实验结果表明,利用本文算法处理后图像的均值（整体亮度）可以提高2倍以上,标准差、熵、峰值信噪比（PSNR）等参数均有5%以上的提升。与其它基于Retinex原理的算法相比,本文提出的算法增强效果显著,具有消除“光晕伪影”现象、抑制噪声、颜色保真和有效地凸显边缘细节信息等能力。     

基于小波与双三次插值的高温锻件红外图像增强方法

提出了一种基于小波变换和双三次插值的图像处理方法,该方法利用离散小波将原图像分解成低频子带与高频子带,然后对原图像及这些子带分别进行双三次插值,同时提出增加一个中间步骤来估算高频子带,即扣除原低分辨率图像和插值后的LL子带中的相同成分而得到不同成分的图像（高频成分）,然后利用不同成分的图像对高频子带LH、HL及HH分别进行校正融合,并通过小波逆变换对这些图像进行融合重构。该方法不仅能够有效地消除图像噪声,改善细节信息,而且能够最大限度地保留边缘信息和增加图像的清晰度。最后运用该方法和传统算法分别对核电主管道高温锻件和小型高温锻件的红外图像进行增强处理,实验结果验证了该方法的可行性及优越性。