Color compensation of multicolor fish images.

Multicolor fluorescence in situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Due to the overlap of excitation and emission spectra and the broad sensitivity of image sensors, the obtained images contain crosstalk between the color channels. The crosstalk complicates both visual and automatic image analysis and may eventually affect the classification accuracy in M-FISH. The removal of crosstalk is possible by finding the color compensation matrix, which quantifies the color spillover between channels. However, there exists no simple method of finding the color compensation matrix from multichannel fluorescence images whose specimens are combinatorially hybridized. In this paper, we present a method of calculating the color compensation matrix for multichannel fluorescence images whose specimens are combinatorially stained.

Feature normalization via expectation maximization and unsupervised nonparametric classification for M-FISH chromosome images.

Multicolor fluorescence in situ hybridization (M-FISH) techniques provide color karyotyping that allows simultaneous analysis of numerical and structural abnormalities of whole human chromosomes. Chromosomes are stained combinatorially in M-FISH. By analyzing the intensity combinations of each pixel, all chromosome pixels in an image are classified. Often, the intensity distributions between different images are found to be considerably different and the difference becomes the source of misclassifications of the pixels. Improved pixel classification accuracy is the most important task to ensure the success of the M-FISH technique. In this paper, we introduce a new feature normalization method for M-FISH images that reduces the difference in the feature distributions among different images using the expectation maximization (EM) algorithm. We also introduce a new unsupervised, nonparametric classification method for M-FISH images. The performance of the classifier is as accurate as the maximum-likelihood classifier, whose accuracy also significantly improved after the EM normalization. We would expect that any classifier will likely produce an improved classification accuracy following the EM normalization. Since the developed classification method does not require training data, it is highly convenient when ground truth does not exist. A significant improvement was achieved on the pixel classification accuracy after the new feature normalization. Indeed, the overall pixel classification accuracy improved by 20% after EM normalization.

Maximum-likelihood decomposition of overlapping and touching M-FISH chromosomes using geometry, size and color information.

Since the birth of chromosome analysis by the aid of computers, building a fully automated chromosome analysis system has been the ultimate goal. Along with many other challenges, automating chromosome classification and segmentation has been one of the major challenges especially due to overlapping and touching chromosomes. In this paper we present a novel decomposition method for overlapping and touching chromosomes in M-FISH images. To overcome the limited success of previous decomposition methods that use partial information about a chromosome cluster, we have incorporated more knowledge about the clusters into a maximum-likelihood frame work. The proposed method evaluates multiple hypotheses based on geometric information, pixel classification results, and chromosome sizes, and a hypothesis that has a maximum-likelihood is chosen as the best decomposition of a given cluster. About 90% of accuracy was obtained for two or three chromosome clusters, which consist about 95% of all clusters with two or more chromosomes.

Subspace-based prototyping and classification of chromosome images.

Chromosomes are essential genomic information carriers. Chromosome classification constitutes an important part of routine clinical and cancer cytogenetics analysis. Cytogeneticists perform visual interpretation of banded chromosome images according to the diagrammatic models of various chromosome types known as the ideograms, which mimic artists' depiction of the chromosomes. In this paper, we present a subspace-based approach for automated prototyping and classification of chromosome images. We show that 1) prototype chromosome images can be quantitatively synthesized from a subspace to objectively represent the chromosome images of a given type or population, and 2) the transformation coefficients (or projected coordinate values of sample chromosomes) in the subspace can be utilized as the extracted feature measurements for classification purposes. We examine in particular the formation of three well-known subspaces, namely the ones derived from principal component analysis (PCA), Fisher's linear discriminant analysis, and the discrete cosine transform (DCT). These subspaces are implemented and evaluated for prototyping two-dimensional (2-D) images and for classification of both 2-D images and one-dimensional profiles of chromosomes. Experimental results show that previously unseen prototype chromosome images of high visual quality can be synthesized using the proposed subspace-based method, and that PCA and the DCT significantly outperform the well-known benchmark technique of weighted density distribution functions in classifying 2-D chromosome images.

Wavelet-based compression of M-FISH images.

Multiplex fluorescence in situ hybridization (M-FISH) is a recently developed technology that enables multi-color chromosome karyotyping for molecular cytogenetic analysis. Each M-FISH image set consists of a number of aligned images of the same chromosome specimen captured at different optical wavelength. This paper presents embedded M-FISH image coding (EMIC), where the foreground objects/chromosomes and the background objects/images are coded separately. We first apply critically sampled integer wavelet transforms to both the foreground and the background. We then use object-based bit-plane coding to compress each object and generate separate embedded bitstreams that allow continuous lossy-to-lossless compression of the foreground and the background. For efficient arithmetic coding of bit planes, we propose a method of designing an optimal context model that specifically exploits the statistical characteristics of M-FISH images in the wavelet domain. Our experiments show that EMIC achieves nearly twice as much compression as Lempel-Ziv-Welch coding. EMIC also performs much better than JPEG-LS and JPEG-2000 for lossless coding. The lossy performance of EMIC is significantly better than that of coding each M-FISH image with JPEG-2000.

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.

Joint segmentation and classification of M-FISH chromosome images.

Automatic segmentation and classification of M-FISH chromosome images are jointly performed using a six-feature, 25-class maximum-likelihood classifier. Preprocessing of the images including background correction and six-channel color compensation method are introduced. A feature transformation method, spherical coordinate transformation, is introduced. High correct classification results are obtained.

Chromosome image enhancement using multiscale differential operators.

Chromosome banding patterns are very important features for karyotyping, based on which cytogenetic diagnosis procedures are conducted. Due to cell culture, staining, and imaging conditions, image enhancement is a desirable preprocessing step before performing chromosome classification. In this paper, we apply a family of differential wavelet transforms (Wang and Lee, 1998), (Wang, 1999) for this purpose. The proposed differential filters facilitate the extraction of multiscale geometric features of chromosome images. Moreover, desirable fast computation can be realized. We study the behavior of both banding edge pattern and noise in the wavelet transform domain. Based on the fact that image geometrical features like edges are correlated across different scales in the wavelet representation, a multiscale point-wise product (MPP) is used to characterize the correlation of the image features in the scale-space. A novel algorithm is proposed for the enhancement of banding patterns in a chromosome image. In order to compare objectively the performance of the proposed algorithm against several existing image-enhancement techniques, a quantitative criteria, the contrast improvement ratio (CIR), has been adopted to evaluate the enhancement results. The experimental results indicate that the proposed method consistently outperforms existing techniques in terms of the CIR measure, as well as in visual effect. The effect of enhancement on cytogenetic diagnosis is further investigated by classification tests conducted prior to and following the chromosome image enhancement. In comparison with conventional techniques, the proposed method leads to better classification results, thereby benefiting the subsequent cytogenetic diagnosis.

Strategies for automated fetal cell screening.

Studies to date have demonstrated fetal sex determination and aneuploidy detection from maternal blood, but a clinical screening technique has not yet emerged. A key limiting factor is the small number of fetal cells, which makes detection specificity and reliability critical. Visual inspection of unsorted or sorted fetal cells is laborious, and cells can be easily missed. Moreover, it is impractical to examine manually all the separated cells. It is highly likely that automation may increase the number of cells inspected, resulting in higher detection sensitivities. Flow and image cytometry are two feasible approaches for automated detection of cells. This review details computerized microscopy (image cytometry) techniques for the automatic detection of fetal cells. Microscopy-based approaches used to identify fetal origin include: (i) immunocytochemical identification of fetal haemoglobin-specific cells (light or fluorescence microscopy); (ii) identification of sex chromosomes and/or aneuploidy using fluorescence in-situ hybridization; and (iii) morphological identification of nucleated red blood cells using light microscopy. The relevant instrumentation, including motorized stages and filters, cameras and digitizer boards are discussed, and software algorithms, including image enhancement, autofocusing, object detection and relocation, and features for operator review and data analysis, are outlined.

Cascaded differential and wavelet compression of chromosome images.

This paper proposes a new method for chromosome image compression based on an important characteristic of these images: the regions of interest (ROIs) to cytogeneticists for evaluation and diagnosis are well determined and segmented. Such information is utilized to advantage in our compression algorithm, which combines lossless compression of chromosome ROIs with lossy-to-lossless coding of the remaining image parts. This is accomplished by first performing a differential operation on chromosome ROIs for decorrelation, followed by critically sampled integer wavelet transforms on these regions and the remaining image parts. The well-known set partitioning in hierarchical trees (SPIHT) (Said and Perlman, 1996) algorithm is modified to generate separate embedded bit streams for both chromosome ROIs and the rest of the image that allow continuous lossy-to-lossless compression of both (although lossless compression of the former is commonly used in practice). Experiments on two sets of sample chromosome spread and karyotype images indicate that the proposed approach significantly outperforms current compression techniques used in commercial karyotyping systems and JPEG-2000 compression, which does not provide the desirable support for lossless compression of arbitrary ROIs.