Chromosome Images Research Articles

Supervised chromosome clustering and image classification

In this paper we propose handwritten signature classification using a supervised chromosome clustering technique. Due to the time variant nature of handwriting of human being, a set of hundred sample handwritten signatures were first collected from the user or individual in form of the same sized grayscale images. These grayscale handwritten signature images will be used as the training set in our classification algorithm. Our proposed algorithm will then decide whether the future incoming handwritten signature of an individual can be a member of the training set or not. In this paper, the distance and similarities play an important role, where the greater the dissimilarity measure or distance of genes, the more dissimilar are the two chromosomes.

Automatic Pattern Extraction and Classification for Chromosome Images

Chromosome image analysis and pattern classification is one of the essential tasks in genetic syndrome diagnoses. An automatic procedure is introduced for chromosome image analysis. The pale-path algorithm is proposed to segment touching and overlapping chromosomes. Medial axis is extracted by the middle point algorithm. Chromosome band is enhanced by the algorithm based on multiscale wavelets Bi-spline, and extracted by average gray profile, gradient profile and shape profile. The multilayer classifier is used to classify the chromosome pattern calculated by weighted density distribution algorithm. Experiment results demonstrate that the algorithms perform well.

A Novel Metric for Bone Marrow Cells Chromosome Pairing

Karyotyping is a set of procedures, in the scope of the cytogenetics, that produces a visual representation of the 46 chromosomes observed during the metaphase step of the cellular division, called mitosis, paired and arranged in decreasing order of size. Automatic pairing of bone marrow cells is a difficult task because these chromosomes appear distorted, overlapped, and their images are usually blurred with undefined edges and low level of detail. In this paper, a new metric is proposed to compare this type of chromosome images toward the design of an automatic pairing algorithm for leukemia diagnostic purposes. Besides the features used in the traditional karyotyping procedures, a new feature, based on mutual information , is proposed to increase the discriminate power of the G-banding pattern dissimilarity between chromosomes and improve the performance of the classifier. The pairing algorithm is formulated as a combinatorial optimization problem where the distances between homologous chromosomes are minimized and the distances between nonhomologous ones are maximized. The optimization task is solved by using an integer programming approach. A new bone marrow chromosome dataset--Lisbon-K1 (LK1) chromosome dataset with 9200 chromosomes---was build for this study. These chromosomes have much lower quality than the classic Copenhagen, Edinburgh, and Philadelphia datasets, and its classification and pairing is therefore more difficult. Experiments using real images from the LK(1) and Grisan et al. datasets based on a leave-one-out cross-validation strategy are performed to test and validate the pairing algorithm.

Integration of cytogenetic and genetic linkage maps of Lotus japonicus, a model plant for legumes

To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.

Karyotype Analysis in Octoploid and Decaploid Wild Strawberries in Fragaria (Rosaceae)

Karyotype analyses have been useful for clarifying phylogenetic and evolutionary relationships among related species and sub-specific taxa. Within the 22 wild species in the genus Fragaria (Rosaceae), karyotype analyses had been restricted to diploid species. The objectives of this research were to determine the chromosomal morphology and analyze the karyotypes of wild octoploid and decaploid species within this genus. Somatic chromosome images of 16 wild genotypes of F. chiloensis, F. virginiana and F. iturupensis were taken at the metaphase stage of mitosis under a light microscope. To evaluate phylogenetic relationships among 12 accessions, karyotypic similarity was examined by cluster analysis. Chromosome numbers of F. chiloensis and F. virginiana genotypes were octoploid (2n=8x=56), except for PI 551462 (2n=8x−2=54), an aneuploid F. chiloensis subsp. pacifica, and PI 551527, a decaploid genotype of F. virginiana subsp. platypetala. Chromosome morphology in F. virginiana subspecies varied more than that in F. chiloensis subspecies. Results of the cluster analyses separated the F. chiloensis and F. virginiana subspecies, except for F. virginiana subsp. grayana PI 616699. The decaploid, F. iturupensis, clustered within the octoploid F. virginiana clade, and was particularly close to F. virginiana subsp. glauca.

Chromosomal organization of the genomes of small-chromosome plants

An effective approach to study the chromosome organization in genomes of plants with small chromosomes and/or with low-informative C-banding patterns was developed in the course of investigation of the karyotypes of cotton plant, camomile, flax, and pea. To increase the resolving power of chromosome analysis, methods were worked out for revealing early replication patterns on chromosomes and for artificial impairment of mitotic chromosome condensation with the use of a DNA intercalator, 9-aminoacridine (9-AMA). To estimate polymorphism of the patterns of C-banding of small chromosomes on preparations obtained with the use of 9-AMA, it is necessary to choose a length interval that must not exceed three average sizes of metaphase chromosomes without the intercalator. The use of 9-AMA increases the resolution of differential C- and OR-banding and the precision of physical chromosome mapping by the FISH method. Of particular importance in studying small chromosomes is optimization of the computer-aided methods used to obtain and process chromosome images. The complex approach developed for analysis of the chromosome organization in plant genomes was used to study the karyotypes of 24 species of the genus Linum L. It permitted their chromosomes to be identified for the first time, and, in addition, B chromosomes were discovered and studied in the karyotypes of the species of the section Syllinum. By similarity of the karyotypes, the studied flax species were distributed in eight groups in agreement with the clusterization of these species according to the results of RAPD analysis performed in parallel. Systematic positions and phylogenetic relationships of the studied flax species were verified. Out results can serve as an important argument in favour of the proposal to develop a special program for sequencing the genome of cultivated flax (L. usitatissimum L.), which is a major representative of small-chromosome species.

Imaging of chromosomes at nanometer-scale resolution using soft X-ray microscope at Ritsumeikan University SR Center

In order to clarify the process of condensation and distribution of a chromosome with 70 nm resolution, process of cell division was observed by soft X-ray microscopy. During prometaphase of mouse fibroblast cell line NIH3T3, each chromosome is clearly visualized at high contrast. Furthermore, thickness of a chromosome is not uniform but varied from 150 nm to 750 nm. This is considered as a condensing step of chromatin fiber occurring in the prometaphase. During metaphase and anaphase, each chromosome was also observed. The chromosomes were thicker than those in prometaphase. This result implies that during metaphase and anaphase the chromosomes condense and become more compact.

Structural Analyses of Chromosomes and Their Constituent Proteins

The importance of chromosome structural study is first described. Then an overview of historical imaging methods that enable us to quantitatively understand chromosome images and structure is given with special reference to the identification of small plant chromosomes and development of their quantitative chromosome maps. A three-dimensional understanding of chromosome distribution within a nucleus answers why the gene-rich regions localize at both ends of chromosomes, especially in the case of species with Rabl orientation. Not only imaging methods but also proteomic approaches are effective in understanding chromosome structure. Over 200 proteins have been identified by proteome analysis of human metaphase chromosomes, and are categorized into four distinct groups according to their nature and localization on chromosomes. These are chromosome coating proteins (CCPs), chromosome peripheral proteins (CPPs), chromosome structural proteins (CSPs), and chromosome fibrous proteins (CFPs). A chromosome four layer model has been developed accordingly. Case studies on individual identified proteins are further described, and the functional similarities of CPPs are exemplified. In addition the controversial roles of CSPs (topoisomerase and condensin), especially for development of higher-order chromosome structure, are discussed. Finally, it is concluded that further advances in chromosome research are necessary to solve an enigma lasting nearly two centuries, that is, why chromosomes retain the same shape in plants and animals.

Enhancement of Multichannel Chromosome Classification Using a Region-Based Classifier and Vector Median Filtering

Multichannel chromosome image acquisition is used for cancer diagnosis and research on genetic disorders. This type of imaging, apart from aiding the cytogeneticist in several ways, facilitates the visual detection of chromosome abnormalities. However, chromosome misclassification errors result from different factors, such as uneven hybridization, spectral overlap among fluors, and biochemical noise. In this paper, we enhance the chromosome classification accuracy by making use of a region Bayes classifier that increases the classification accuracy when compared to the already developed pixel-by-pixel classifier and by incorporating the vector median filtering approach for filtering of the image. The method is evaluated using a publicly available database that contains 183 six-channel chromosome sets of images. The overall improvement on the chromosome classification accuracy is 9.99%, compared to the pixel-by-pixel classifier without filtering. This improvement in the chromosome classification accuracy would allow subtle deoxyribonucleic acid abnormalities to be identified easily. The efficiency of the method might further improve by using features extracted from each region and a more sophisticated classifier.

Structural analysis of human chromosomes by atomic force and light microscopy in relation to the distribution of topoisomerase II.ALPHA.

The relationship between the higher-order structure of human metaphase chromosomes and the distribution of topoisomerase IIα was analyzed by a comparison of atomic force microscope (AFM) and fluorescence microscope images of the same chromosome. AFM imaging of chromosomes in liquid revealed the presence of alternating ridges and grooves on the surfaces of the sister chromatids. In contrast, the fluorescence image of the chromosomes stained with the anti-topoisomerase IIα antibody showed that the fluorescence intensity of topoisomerase IIα was not uniform and that there were alternating strong and weak spots along the chromosome axes. A comparison of the AFM image with a fluorescence microscope image of the same chromosome further demonstrated that ridges and grooves corresponded to strong and weak fluorescence intensities of topoisomerase IIα, respectively. These findings suggest that the distribution of topoisomerase IIα has a close connection with the higher-order structure of human metaphase chromosomes.

Congenital Optic Disc Coloboma Associated With Right Seventh and Eighth Cranial Nerve Palsy

Purpose: To report a case of bilateral congenital optic disc coloboma associated with the right seventh and eighth cranial nerve palsy. Case Summary: A female neonate with right facial palsy (seventh cranial nerve palsy) and right earlobe hypoplasia was referred for examination for retinopathy of prematurity (ROP). Bilateral optic disc coloboma and peripapillary choroidal defect was detected on the fundus examination and the anterior segment examination revealed no specific findings. On the otolaryngologic examination, laryngomalacia and floppy epiglottis were observed and left otitis media and mastoiditis were noted on the temporal bone computed tomography (CT). On the auditory brain stem response (ABR), right electro-potential was not detected and right cochlear nerve palsy (eighth cranial nerve palsy) was diagnosed. Further chromosomal analysis and brain magnetic resonance imaging (MRI) revealed no abnormal findings. However, on echocardiography, an atrial septal defect was detected and on upper gastrointestinal series, gastroesophageal reflux disease (GERD) was diagnosed. Conclusions: Congenital optic disc coloboma is frequently accompanied by other congenital deformities or abnormalities, and therefore, systemic examinations and tests to detect associated findings are required.

Karyotype Analysis in Wild Diploid, Tetraploid and Hexaploid Strawberries, Fragaria (Rosaceae)

The strawberry, genus Fragaria (Rosaceae), has a basic chromosome of x=7, and is comprised of 20 wild species having an euploid series from diploid (2n=2x=14) through decaploid (2n=10x=70). Few karyotypes of species in this genus have been reported. The objective of this research was to determine the chromosomal morphology and karyotype analysis of wild diploid, tetraploid and hexaploid Fragaria species. Somatic chromosome images of 20 genotypes of diploids, tetraploids, and hexaploids were taken at the metaphase stage of mitosis under a light microscope. Karyotype analysis was performed in 17 accessions. The phylogenetic relationships between species were constructed using cluster analysis based on karyotypic similarity. Somatic chromosome numbers of wild diploid, tetraploid and hexaploid species were 2n=2x=14, 2n=4x=28 and 2n=6x=42. Chromosome morphology in wild diploid species had greater uniformity than that in the tetraploid species. Results of the cluster analysis showed that the diploid and tetraploid species reside in separate clades, with one exception. Fragaria tibetica, a tetraploid, clustered with the diploid species clade. The hexaploid, F. moschata, clustered with the tetraploid species clade.

Improvement of staining method for observation of mitotic chromosomes in octoploid strawberry plants

An important strategy to conduct intentional breeding of octoploid strawberry plants is to recognize the functions of every chromosome. To do so, a methodology must be developed to distinguish chromosomes one by one. We reported the possibility of distinguishing chromosomes using light microscopy when somatic cells of octoploid strawberry plants were stained using ordinary methods with lacto-propionic orcein (LPO). However, karyotype analysis of octoploid strawberry plants required clearer chromosome images. This study obtained clearer chromosome images of octoploid Fragaria × ananassa and F. chiloensis plants. Three staining methods were examined: 60% acetic acid (AA) alone, 1.5% LPO alone, and two-step treatments with 60% AA and 1.5% LPO. Collected root tips of the plants were placed in 2 mM 8-hydroxyquinoline solution for 1 h and were subsequently stored at 4 °C for 15 h. The samples were then fixed in a 3:1 absolute alcohol: glacial acetic acid solution for 40 min, followed by mixture with 1N HCl solutions at room temperature for 2 h and then at 60 °C for 10 min. For separate staining using 60% AA and 1.5% LPO, the root tip was expelled on the glass slide with a drop of each solution for a few minutes to stain the chromosomes. For the two-step staining method, the samples stained with 60% AA were frozen at −80 °C for at least 5 min. The cover slip was removed using a razor blade. Subsequently, the specimens were air-dried and stained with the 1.5% LPO for 3 min. Digital images of chromosomes were obtained using light microscopy. Samples of the two-step staining method produced the clearest chromosome images in both F. × ananassa and F. chiloensis. Furthermore, the greatest color difference between the chromosomes and the cytoplasm was obtained from images of the two-step staining method among the three staining methods. These results demonstrate that the two-step staining method is useful for chromosome counting and karyotype analysis in strawberry plants.

Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

Although the formation of 30-nm chromatin fibers is thought to be the most basic event of chromatin compaction, it remains controversial because high-resolution imaging of chromatin in living eukaryotic cells had not been possible until now. Cryo-electron microscopy of vitreous sections is a relatively new technique, which enables direct high-resolution observation of the cell structures in a close-to-native state. We used cryo-electron microscopy and image processing to further investigate the presence of 30-nm chromatin fibers in human mitotic chromosomes. HeLa S3 cells were vitrified by high-pressure freezing, thin-sectioned, and then imaged under the cryo-electron microscope without any further chemical treatment or staining. For an unambiguous interpretation of the images, the effects of the contrast transfer function were computationally corrected. The mitotic chromosomes of the HeLa S3 cells appeared as compact structures with a homogeneous grainy texture, in which there were no visible 30-nm fibers. Power spectra of the chromosome images also gave no indication of 30-nm chromatin folding. These results, together with our observations of the effects of chromosome swelling, strongly suggest that, within the bulk of compact metaphase chromosomes, the nucleosomal fiber does not undergo 30-nm folding, but exists in a highly disordered and interdigitated state, which is, on the local scale, comparable with a polymer melt.

Caulobacter requires a dedicated mechanism to initiate chromosome segregation

Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show that Caulobacter crescentus makes use of and requires a dedicated mechanism to initiate chromosome segregation. Caulobacter has a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin and parS moves rapidly to the opposite pole. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. We use chromosomal inversions and in vivo time-lapse imaging to show that parS is the Caulobacter site of force exertion, independent of its position in the chromosome. When parS is moved farther from the origin, the cell waits for parS to be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation in Caulobacter cannot occur unless a dedicated parS guiding mechanism initiates movement.

Development and Assessment of an Integrated Computer-Aided Detection Scheme for Digital Microscopic Images of Metaphase Chromosomes.

The authors developed an integrated computer-aided detection (CAD) scheme for detecting and classifying metaphase chromosomes as well as assessing its performance and robustness. This scheme includes an automatic metaphase-finding module and a karyotyping module and it was applied to a testing database with 200 digital microscopic images. The automatic metaphase-finding module detects analyzable metaphase cells using a feature-based artificial neural network (ANN). The ANN-generated outputs are analyzed by a receiver operating characteristics (ROC) method and an area under the ROC curve is 0.966. Then, the automatic karyotyping module classifies individual chromosomes of this cell into 24 types. In this module, a two-layer decision tree-based classifier with eight ANNs established in its connection nodes was optimized by a genetic algorithm. Chromosomes are first classified into seven groups by the ANN in the first layer. The chromosomes in these groups are then separately classified by seven ANNs into 24 types in the second layer. The classification accuracy is 94.5% in the first layer. Six ANNs achieved the accuracy above 95% and only one had lessened performance (80.6%) in the second layer. The overall classification accuracy is 91.5% as compared to 86.7% in the previous study using two independent datasets randomly acquired from our genetic laboratory. The results demonstrate that our automated scheme achieves high and robust performance in identification and classification of metaphase chromosomes.

High-speed AFM of human chromosomes in liquid

Further developments of the previously reported high-speed contact-mode AFM aredescribed. The technique is applied to the imaging of human chromosomes at video rateboth in air and in water. These are the largest structures to have been imaged withhigh-speed AFM and the first imaging in liquid to be reported. A possible mechanism thatallows such high-speed contact-mode imaging without significant damage to the sample isdiscussed in the context of the velocity dependence of the measured lateral force on theAFM tip.

Nanotomography of Cell Surfaces with Evanescent Fields

The technique of variable-angle total internal reflection fluorescence microscopy (TIRFM) and its application to nanotomography of cell surfaces are described. Present applications include (1) 3D imaging of chromosomes in their metaphase to demonstrate axial resolution in the nanometre range, (2) measurements of cell-substrate topology, which upon cholesterol depletion shows some loosening of cell-substrate contacts, and (3) measurements of cell topology upon photodynamic therapy (PDT), which demonstrate cell swelling and maintenance of focal contacts. The potential of the method for in vitro diagnostics, but also some requirements and limitations are discussed.

A Multichannel Watershed-Based Segmentation Method for Multispectral Chromosome Classification

Multiplex fluorescent in situ hybridization (M-FISH) is a recently developed chromosome imaging technique where each chromosome class appears to have a distinct color. This technique not only facilitates the detection of subtle chromosomal aberrations but also makes the analysis of chromosome images easier; both for human inspection and computerized analysis. In this paper, a novel method for segmentation and classification of M-FISH chromosome images is presented. The segmentation is based on the multichannel watershed transform in order to define regions of similar spatial and spectral characteristics. Then, a Bayes classifier, task-specific on region classification, is applied. Our method consists of four basic steps: 1) computation of the gradient magnitude of the image, 2) application of the watershed transform to decompose the image into a set of homogenous regions, 3) classification of each region, and 4) merging of similar adjacent regions. The method is evaluated using a publicly available chromosome image database and the obtained overall accuracy is 82.4%. By introducing the classification of each watershed region, the proposed method achieves substantially better results compared to other methods at a lower computational cost. The combination of the multichannel segmentation and the region-based classification is found to improve the overall classification accuracy compared to pixel-by-pixel approaches.

Atomic force microscopy for imaging human metaphase chromosomes

The present study introduces the principle of atomic force microscopy (AFM) and reviews our results of human metaphase chromosomes obtained by AFM. AFM imaging of the chromosomes revealed that the chromatid arm was not uniform in structure but had ridges and grooves along its length, which was most prominent in the late metaphase. The arrangement of these ridges and grooves was roughly symmetrical with the counterpart of the paired sister chromatids. AFM imaging of banded chromosomes also showed that the ridges and grooves were related to the G/Q-positive and G/Q-negative bands, respectively. At high magnification, the chromatid was seen to be produced by the compaction of highly twisted chromatin fiber loops, and its compaction tended to be stronger in the ridged regions of the chromosomes than in the grooved regions. Our AFM studies also showed the presence of catenation of chromatin fibers between the ridged portions of the chromatid in the late metaphase. Thus, AFM is useful for obtaining the three-dimensional surface topography not only in ambient conditions but also in physiological liquid conditions, and is expected to be an attractive tool for investigating the structure of chromosomes.