Signal to noise analysis of multiple color fluorescence imaging microscopy

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.     

Normalization of multicolor fluorescence in situ hybridization (M-FISH) images for improving color karyotyping.

BACKGROUND: Multiplex or multicolor fluorescence in situ hybridization (M-FISH) is a recently developed cytogenetic technique for cancer diagnosis and research on genetic disorders. By simultaneously viewing the multiply labeled specimens in different color channels, M-FISH facilitates the detection of subtle chromosomal aberrations. The success of this technique largely depends on the accuracy of pixel classification (color karyotyping). Improvements in classifier performance would allow the elucidation of more complex and more subtle chromosomal rearrangements. Normalization of M-FISH images has a significant effect on the accuracy of classification. In particular, misalignment or misregistration across multiple channels seriously affects classification accuracy. Image normalization, including automated registration, must be done before pixel classification. METHODS AND RESULTS: We studied several image normalization approaches that affect image classification. In particular, we developed an automated registration technique to correct misalignment across the different fluor images (caused by chromatic aberration and other factors). This new registration algorithm is based on wavelets and spline approximations that have computational advantages and improved accuracy. To evaluate the performance improvement brought about by these data normalization approaches, we used the downstream pixel classification accuracy as a measurement. A Bayesian classifier assumed that each of 24 chromosome classes had a normal probability distribution. The effects that this registration and other normalization steps have on subsequent classification accuracy were evaluated on a comprehensive M-FISH database established by Advanced Digital Imaging Research (http://www.adires.com/05/Project/MFISH_DB/MFISH_DB.shtml). CONCLUSIONS: Pixel misclassification errors result from different factors. These include uneven hybridization, spectral overlap among fluors, and image misregistration. Effective preprocessing of M-FISH images can decrease the effects of those factors and thereby increase pixel classification accuracy. The data normalization steps described in this report, such as image registration and background flattening, can significantly improve subsequent classification accuracy. An improved classifier in turn would allow subtle DNA rearrangements to be identified in genetic diagnosis and cancer research.     

Towards many colors in FISH on 3D-preserved interphase nuclei

The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested.     

Multiplex-FISH for pre- and postnatal diagnostic applications.

For >3 decades, Giemsa banding of metaphase chromosomes has been the standard karyotypic analysis for pre- and postnatal diagnostic applications. However, marker chromosomes or structural abnormalities are often encountered that cannot be deciphered by G-banding alone. Here we describe the use of multiplex-FISH (M-FISH), which allows the visualization of the 22 human autosomes and the 2 sex chromosomes, in 24 different colors. By M-FISH, the euchromatin in marker chromosomes could be readily identified. In cases of structural abnormalities, M-FISH identified translocations and insertions or demonstrated that the rearranged chromosome did not contain DNA material from another chromosome. In these cases, deleted or duplicated regions were discerned either by chromosome-specific multicolor bar codes or by comparative genomic hybridization. In addition, M-FISH was able to identify cryptic abnormalities in patients with a normal G-karyotype. In summary, M-FISH is a reliable tool for diagnostic applications, and results can be obtained in 相似文献   

An improved sparse representation model with structural information for Multicolour Fluorescence <Emphasis Type="Italic">In-Situ</Emphasis> Hybridization (M-FISH) image classification

Background

Multicolour Fluorescence In-Situ Hybridization (M-FISH) images are employed for detecting chromosomal abnormalities such as chromosomal translocations, deletions, duplication and inversions. This technique uses mixed colours of fluorochromes to paint the whole chromosomes for rapid detection of chromosome rearrangements. The M-FISH data sets used in our research are obtained from microscopic scanning of a metaphase cell labelled with five different fluorochromes and a DAPI staining. The reliability of the technique lies in accurate classification of chromosomes (24 classes for male and 23 classes for female) from M-FISH images. However, due to imaging noise, mis-alignment between multiple channels and many other imaging problems, there is always a classification error, leading to wrong detection of chromosomal abnormalities. Therefore, how to accurately classify different types of chromosomes from M-FISH images becomes a challenging problem.

Methods

This paper presents a novel sparse representation model considering structural information for the classification of M-FISH images. In our previous work a sparse representation based classification model was proposed. This model employed only individual pixel information for the classification. With the structural information of neighbouring pixels as well as the information of themselves simultaneously, the novel approach extended the previous one to the regional case. Based on Orthogonal Matching Pursuit (OMP), we developed simultaneous OMP algorithm (SOMP) to derive an efficient solution of the improved sparse representation model by incorporating the structural information.

Results

The p-value of two models shows that the newly proposed model incorporating the structural information is significantly superior to our previous one. In addition, we evaluated the effect of several parameters, such as sparsity level, neighbourhood size, and training sample size, on the of the classification accuracy.

Conclusions

The comparison with our previously used sparse model demonstrates that the improved sparse representation model is more effective than the previous one on the classification of the chromosome abnormalities.

     

New concepts to improve resolution and sensitivity of molecular cytogenetic diagnostics by multicolor fluorescence in situ hybridization

BACKGROUND: Routine application of multicolor fluorescence in situ hybridization (M-FISH) technology for molecular cytogenetic diagnostics has been hampered by several technical limitations. First, when using chromosome-specific painting probes, there is a limit in cytogenetic resolution of approximately 2-3 Mb, which can mask hidden structural abnormalities that have a significant clinical effect. Second, using whole chromosome painting probes, intrachromosomal rearrangements cannot be detected and the exact localization of breakpoints is often not possible. METHODS: We suggest the use of multiplex-labeled region or locus- specific probes in combination with an optimal probe design to improve the sensitivity and resolution of the M-FISH technology. To allow the application of this assay in routine diagnostics, we developed a multipurpose image analysis system. RESULTS: goldFISH was applied to the study of cryptic translocations in mental retardation patients and to the study of high-resolution breakpoint mapping in non-small cell lung cancer patients. For an individual with mental retardation, who had an apparently normal karyotype by G-banding, we detected an unbalanced translocation involving chromosomes 2 and 7. CONCLUSIONS: In combination with optimally designed probe kits, goldFISH overcomes most of the present limitations of the M-FISH technology and results in virtually 100% reliability for detecting interchromosomal and intrachromosomal rearrangements.     

Spectral karyotyping analysis of human and mouse chromosomes

Classical banding methods provide basic information about the identities and structures of chromosomes on the basis of their unique banding patterns. Spectral karyotyping (SKY), and the related multiplex fluorescence in situ hybridization (M-FISH), are chromosome-specific multicolor FISH techniques that augment cytogenetic evaluations of malignant disease by providing additional information and improved characterization of aberrant chromosomes that contain DNA sequences not identifiable using conventional banding methods. SKY is based on cohybridization of combinatorially labeled chromosome-painting probes with unique fluorochrome signatures onto human or mouse metaphase chromosome preparations. Image acquisition and analysis use a specialized imaging system, combining Sagnac interferometer and CCD camera images to reconstruct spectral information at each pixel. Here we present a protocol for SKY analysis using commercially available SkyPaint probes, including procedures for metaphase chromosome preparation, slide pretreatment and probe hybridization and detection. SKY analysis requires approximately 6 d.     

Molecular cytogenetic characterization of eight small supernumerary marker chromosomes originating from chromosomes 2, 4, 8,18, and 21 in three patients

Small supernumerary marker chromosomes (sSMCs) are a morphologically heterogeneous group of additional structurally abnormal chromosomes that cannot be identified unambiguously by conventional banding techniques alone. Molecular cytogenetic methods enable detailed characterization of sSMCs; however, in many cases interpretation of their clinical significance is problematic. The aim of our study was to characterize precisely sSMCs identified in three patients with dysmorphic features, psychomotor retardation and multiple congenital anomalies. We also attempted to correlate the patients' genotypes with phenotypes by inclusion of data from the literature. The sSMCs were initially detected by G-banding analysis in peripheral blood lymphocytes in these patients and were subsequently characterized using multicolor fluorescence in situ hybridization (M-FISH), (sub)centromere-specific multicolor FISH (cenM-FISH, subcenM-FISH), and multicolor banding (MCB) techniques. Additionally, the sSMCs in two patients were also studied by hybridization to whole-genome bacterial artificial chromosome (BAC) arrays (array-CGH) to map the breakpoints on a single BAC clone level. In all three patients, the chromosome origin, structure, and euchromatin content of the sSMCs were determined. In patient RS, only a neocentric r(2)(q35q36) was identified. It is a second neocentric sSMC(2) in the literature and the first marker chromosome derived from the terminal part of 2q. In the other two patients, two sSMCs were found, as M-FISH detected additional sSMCs that could not be characterized in G-banding analysis. In patient MK, each of four cell lines contained der(4)(:p11.1-->q12:) accompanied by a sSMC(18): r(18)(:p11.2-->q11.1::p11.2-->q11.1:), inv dup(18)(:p11.1-->q11.1::q11.1-->p11.1:), or der(18) (:p11.2-->q11.1::q11.1-->p11.1:). In patient NP, with clinical features of trisomy 8p, three sSMCs were characterized: r(8)(:p12-->q11.1::q11.1-->p21:) der(8) (:p11.22-->q11.1::q11.1-->p21::p21-->p11.22:) and der(21)(:p11.1-->q21.3:). The BAC array results confirmed the molecular cytogenetic results and refined the breakpoints to the single BAC clone resolution. However, the complex mosaic structure of the marker chromosomes derived from chromosomes 8 and 18 could only be identified by molecular cytogenetic methods. This study confirms the usefulness of multicolor FISH combined with whole-genome arrays for comprehensive analyses of marker chromosomes.     

Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ER alpha cells: bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridization

We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T-antigen (Harris et al. [1995a] J. Bone. Miner. Res. 10:178-1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M-FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1-2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2-3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non-transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro.     

Improvement of signal-to-noise ratio in electroantennogram responses using multiple insect antennae

Using an array of insect antennae connected in series or in parallel, electroantennogram (EAG) responses and noise levels were investigated in an attempt to improve signal-to-noise (S/N) ratio and sensitivity. Both the EAG response amplitude and noise level increased when the antennae of male Helicoverpa zea (Lepidoptera: Noctuidae) were connected in series. Due to lower relative increase in noise level than EAG amplitude as the number of antennae increased, the S/N ratio was also significantly improved by the serial connection. As a result the sensitivity of EAG was improved by the serial connection, which showed ca. ten-fold improvement in the threshold detection levels compared with a single antenna when four antennae were connected in series. In contrast to the serial connection, there were no differences in EAG amplitudes and overall noise levels when different numbers of antennae were connected in parallel. When only large-amplitude noise was taken into account, however, the S/N ratio was somewhat improved by the parallel connection. The frequency of overall noise remained at the same level both in the serial and in the parallel connection. However, the frequency of the large-amplitude noise increased in serial connection but decreased in parallel connection. The present study clearly indicates that both the sensitivity and S/N ratio of the EAG biosensor could be significantly improved by using the multiple antennal connections.     

Resolution of complex fluorescence spectra recorded on single unpigmented living cells using a computerised method

The identification and quantification of fluorescent compounds in a complex fluorescence spectra are always difficult, especially in the case of low signal:noise ratio. We propose a computerised method that allows the resolution of low light level complex fluorescence spectra into its components. Based on a linear combination of N possible characteristic fluorescence spectra, and using N weighting functions, this method allows the integration of fluorescence intensities over the entire fluorescence spectra and the generation of n equations with N unknowns. The compounds that participate in complex fluorescence spectra are identified and quantified. Because fluorescence intensities can be integrated we can resolve complex fluorescence spectra presenting a low signal:noise ratio. The reliability and sensitivity of our method are shown through examples of resolution of complex intracellular fluorescence of single living cells pretreated with benzo(a)pyrene. Depending on the cell type and treatment, two, four, or five components can be identified in the complex fluorescence spectra.     

广东连州自然分布的3种植物体内及根际环境中稳定性碳和氮同位素的分析

以广东连州自然分布的3种国家重点保护野生植物南方红豆杉〔Taxus chinensis var.mairei(Lemée et Lévl.)Cheng et L.K.Fu〕、半枫荷(Semiliquidambar cathayensis Chang)和金荞麦〔Fagopyrum dibotrys(D.Don)Hara〕为研究对象,分析了根、茎和叶片及根际土壤和岩石的C、N含量和C:N比以及δ13 C和δ15 N值的差异;在此基础上,通过δ13 C和δ15 N值的散点图比较了3种植物生态位的差异.结果表明:在同种植物中,根、茎和叶片的C和N含量及C:N比总体高于根际土壤和岩石,其中,叶片中C和N含量均最高,茎的C:N比最高;而根际土壤和岩石的δ13 C和δ15 N值总体高于根、茎和叶片.在供试的3种植物间,根际土壤和岩石中C和N含量总体上无明显差异,但根、茎和叶片中C和N含量以及根、茎和叶片及根际土壤和岩石的C:N比、δ13 C和δ15 N值均有一定差异;其中,金荞麦根中C含量显著(P<0.05)低于南方红豆杉和半枫荷,其根、茎和叶片中N含量和δ15 N值均极显著(P<0.01)高于后二者,其根、茎和叶片的C:N比和δ13 C值均极显著低于后二者,其根际土壤和岩石的C:N比和δ13 C值总体上也低于后二者;南方红豆杉和半枫荷的叶片中C和N含量以及茎和叶片的δ13 C值、根际土壤和岩石的δ13 C和δ15 N值均存在显著差异,但二者的整体差异相对较小.从散点图上看,金荞麦的生态位远离南方红豆杉和半枫荷,而后二者的生态位有交集.综合分析结果显示:草本植物金荞麦与木本植物南方红豆杉和半枫荷的C和N含量以及δ13 C和δ15 N值的差异不仅与植物自身的生活型有关,而且与各自生境中的光照和土壤因子等相关.另外,供试3种植物的根、茎和叶片的δ13 C值变幅为-31.69‰~-26.46‰,符合C3植物的δ13 C值范畴.     

Characterisation of the alpha 1-protease inhibitor system in Thoroughbred horse plasma by horizontal two-dimensional (ISO-DALT) electrophoresis. 2. Protease inhibition

The protease inhibitory spectra of the eight homozygous Thoroughbred Pi types against trypsin, elastase and chymotrypsin have been determined. The alpha 1-protease inhibitor proteins exhibit three classes of inhibitory specificity towards these enzymes. The Pi types F, I, N and U exhibit class I (trypsin, elastase and chymotrypsin) and class II (trypsin and elastase) types of inhibition and fit Juneja et al.'s (1979) classification of two separate genetic systems Pi 1 and Pi 2 based on differences in the inhibitory spectra against trypsin and chymotrypsin. The remaining four Pi types are exceptions to Juneja et al.'s (1979) classification. Types G, L, S1 and S2 possess class I but not class II proteins. A third class of proteins (class III) which exclusively inhibit chymotrypsin was detected in all eight protease inhibitor types. Type G is well represented by class III proteins because two of the three major proteins of the ISO-DALT pattern inhibit only chymotrypsin and is thus an exception to Juneja et al.'s (1979) classification.     

Noninvasive tumor volume measurement using magnetic resonance (MR) imaging and an open sided saddle coil

A magnetic resonance (MR) imaging scanner operated at 0.5 T with a specially constructed receiving coil was used to measure volumes of primary spontaneous tumors in rats and guinea pigs. The coil was used to improve the signal to noise ratio (S/N) of the MR images of tumors in these small animals. The tumor volume was determined by the summation of the volume of contiguous slices or ellipsoid approximation. The accuracy of the volume measurement was better when the numerical integration was used in calculating the slice volume. The open sided saddle (OSS) coil used as the receiving coil gave better S/N than that of the standard head coil.     

Analysis of machine-selected cells with an image analysis system in normal and abnormal cervical specimens

An analysis has been performed of visual diagnostic criteria used in cervical cytology applied to machine selected cells in relation to automated classification based on variables, which can be recorded in an image system with automated cell search and segmentation, feature extraction and classification. A 98% accuracy could be obtained with the choice of the most ideal statistical methods for discrimination and the use of the most powerful variables recorded in the image system when compared with consensus of the visual diagnoses based on established cytological criteria for diagnosis of cancer and precancer of the cervix uteri. The most powerful discriminatory variables in the image system (of 17 recorded) for discrimination between normal and abnormal epithelial cells were, in addition to nuclear extinction, cytoplasmic extinction and cytoplasmic shape. It is concluded that the visual classification of cervical cells is highly accurate with experienced observers and that imaging microscopes can be trained to nearly equal this accuracy with appropriate statistical methods of discrimination. The problem of creating fully automated systems, however, also requires the inclusion of even more effective discriminatory variables and also the solution of such problems as automatic cell search, segmentation, artifact rejection, feature extraction, classification and electronic stability in order to become cost-effective.     

Plant light interception can be explained via computed tomography scanning: demonstration with pyramidal cedar (Thuja occidentalis, Fastigiata)

BACKGROUND AND AIMS: Light interception by the leaf canopy is a key aspect of plant photosynthesis, which helps mitigate the greenhouse effect via atmospheric CO(2) recycling. The relationship between plant light interception and leaf area was traditionally modelled with the Beer-Lambert law, until the spatial distribution of leaves was incorporated through the fractal dimension of leafless plant structure photographed from the side allowing maximum appearance of branches and petioles. However, photographs of leafless plants are two-dimensional projections of three-dimensional structures, and sampled plants were cut at the stem base before leaf blades were detached manually, so canopy development could not be followed for individual plants. Therefore, a new measurement and modelling approach were developed to explain plant light interception more completely and precisely, based on appropriate processing of computed tomography (CT) scanning data collected for developing canopies. METHODS: Three-dimensional images of canopies were constructed from CT scanning data. Leaf volumes (LV) were evaluated from complete canopy images, and fractal dimensions (FD) were estimated from skeletonized leafless images. The experimental plant species is pyramidal cedar (Thuja occidentalis, Fastigiata). KEY RESULTS: The three-dimensional version of the Beer-Lambert law based on FD alone provided a much better explanation of plant light interception (R(2) = 0.858) than those using the product LV*FD (0.589) or LV alone (0.548). While values of all three regressors were found to increase over time, FD in the Beer-Lambert law followed the increase in light interception the most closely. The delayed increase of LV reflected the appearance of new leaves only after branches had lengthened and ramified. CONCLUSIONS: The very strong correlation obtained with FD demonstrates that CT scanning data contain fundamental information about the canopy architecture geometry. The model can be used to identify crops and plantation trees with improved light interception and productivity.     

A mathematical programming approach for gene selection and tissue classification

MOTIVATION: Extracting useful information from expression levels of thousands of genes generated with microarray technology needs a variety of analytical techniques. Mathematical programming approaches for classification analysis outperform parametric methods when the data depart from assumptions underlying these methods. Therefore, a mathematical programming approach is developed for gene selection and tissue classification using gene expression profiles. RESULTS: A new mixed integer programming model is formulated for this purpose. The mixed integer programming model simultaneously selects genes and constructs a classification model to classify two groups of tissue samples as accurately as possible. Very encouraging results were obtained with two data sets from the literature as examples. These results show that the mathematical programming approach can rival or outperform traditional classification methods.