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SummaryEssential features of cancer tissue cellular heterogeneity such as negatively selected genome topologies, sub-clonal mutation patterns and genome replication states can only effectively be studied by sequencing single-cell genomes at scale and high fidelity. Using an amplification-free single-cell genome sequencing approach implemented on commodity hardware (DLP+) coupled with a cloud-based computational platform, we define a resource of 40,000 single-cell genomes characterized by their genome states, across a wide range of tissue types and conditions. We show that shallow sequencing across thousands of genomes permits reconstruction of clonal genomes to single nucleotide resolution through aggregation analysis of cells sharing higher order genome structure. From large-scale population analysis over thousands of cells, we identify rare cells exhibiting mitotic mis-segregation of whole chromosomes. We observe that tissue derived scWGS libraries exhibit lower rates of whole chromosome anueploidy than cell lines, and loss of p53 results in a shift in event type, but not overall prevalence in breast epithelium. Finally, we demonstrate that the replication states of genomes can be identified, allowing the number and proportion of replicating cells, as well as the chromosomal pattern of replication to be unambiguously identified in single-cell genome sequencing experiments. The combined annotated resource and approach provide a re-implementable large scale platform for studying lineages and tissue heterogeneity.

Summary Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations., Graphical Abstract, Highlights • Scaled method and resource of > 50K single-cell whole genomes from diverse cell types • Clonal merging can resolve clone specific mutations to single-nucleotide level • Image analysis of single cells permits correlation of morphology and genome features • Clonal replication states and rare aneuploidy patterns of single cells measured, A high-throughput method for amplication-free single-cell whole-genome sequencing can be scaled up to analyze tens of thousands of cells from different tissues and clinical sample types and identifies replication states, aneuploidies, and subclonal mutations.

Purpose Evaluation of the relationships existing among 3 histologic types of urothelial tumors, chromosomal instability, and telomere length. Patients and methods We examined 37 consecutive cases of papillary urothelial neoplasm, from which 26 (70.3%) were suitable for karyotype analysis, comprising 7 papillary urothelial neoplasms of low malignant potential (PUNLMPs), 10 low-grade papillary urothelial carcinomas (PUCs), and 9 high-grade PUCs. We performed karyotype and anaphase bridge analyses, and measured telomere lengths by quantitative fluorescence in situ hybridization. Results PUNLMPs were always diploid and had anaphase bridges. Low-grade PUCs showed diploidy ( n = 2), hypoploidy ( n = 4) and polyploidy ( n = 4), and high-grade PUCs showed diploidy ( n = 1) and polyploidy ( n = 8); both had anaphase bridges. The incidence of anaphase bridges did not differ significantly between PUNLMPs and high-grade PUCs ( P = 0.105). The telomere lengths of PUNLMP, low-grade PUC, and high-grade PUC, expressed as mean telomere fluorescence units (TFU)±SD, were 7906±3197, 4893±1567, and 3299±1406, respectively. The differences among the 3 groups were significant. However, 42.9% of the PUNLMPs had shorter telomeres than the mean value for low-grade PUCs, and 30.0% of the low-grade PUCs had shorter telomeres than those for high-grade PUCs. There was an inverse correlation between telomere length and the incidence of anaphase bridges. Conclusions PUNLMP appears to progress to low-grade PUC and high-grade PUC in association with telomere shortening and chromosomal instability. Our data suggest that critically shortened telomeres cause chromosomal instability during progression of papillary urothelial neoplasms.

The human epidermal growth factor receptor 2 (HER2) proto-oncogene plays an important role in the development and progression of breast and gastric cancer. Monitoring of the HER2 status and treatment with trastuzumab was performed initially in breast cancer, and subsequently in gastric cancer. However, the HER2 status of thyroid cancer remains unexplored. Telomere alteration and telomerase activity have been observed in most human cancers and are known to be a feature of malignancy. The aims of this study were to clarify the HER2 status of thyroid cancer and to examine any correlations to various characteristics of malignancy. We investigated 69 cases of differentiated thyroid cancers with reference to: i) telomere length as measured using tissue quantitative fluorescence in situ hybridization (Q-FISH), ii) expression of human telomerase reverse transcriptase (hTERT) as determined by immunohistochemistry (IHC), and iii) overexpression of the HER2 protein as determined by IHC and amplification of the HER2 gene as determined by fluorescence in situ hybridization (FISH). The telomeres of thyroid cancers, especially follicular carcinomas, were significantly shorter compared to those of adjacent normal tissues. Positivity for hTERT expression and HER2 amplification were observed in approximately 70 and 22% of thyroid cancers, respectively. Our data demonstrated that telomeres in HER2-positive cancers were significantly shorter compared to those in HER2-negative cancers. These results suggest that highly malignant differentiated thyroid cancer can be detected by monitoring HER2 status and telomere shortening, and that trastuzumab therapy may be effective for refractory thyroid cancer.

We have reported telomere attrition in β and α cells of the pancreas in elderly patients with type 2 diabetes, but it has not been explored how the telomere lengths of these islet cells change according to age in normal subjects. To examine the telomere lengths of β and α cells in individuals without diabetes across a wide range of ages, we conducted measurement of the telomere lengths of human pancreatic β and α cells obtained from 104 autopsied subjects without diabetes ranging in age from 0 to 100 years. As an index of telomere lengths, the normalized telomere-centromere ratio (NTCR) was determined for β (NTCRβ) and α (NTCRα) cells by quantitative fluorescence in situ hybridization (Q-FISH). We found NTCRβ and NTCRα showed almost the same levels and both decreased according to age (p

Liver transplantation for biliary atresia is indicated whenever a Kasai portoenterostomy is considered unfeasible. However, the timing of liver transplantation in biliary atresia has not been precisely defined. Excessive shortening of hepatocellular telomeres may occur in patients with biliary atresia, and therefore, telomere length could be a predictor of hepatocellular reserve capacity. Hepatic tissues were obtained from 20 patients with biliary atresia who underwent LT and 10 age-matched autopsied individuals (mean age, 1.7 and 1.2 years, respectively). Telomere lengths were measured by Southern blotting and quantitative fluorescence in situ hybridization using the normalized telomere-centromere ratio. The correlation between the normalized telomere-centromere ratio for the hepatocytes in biliary atresia and the pediatric end-stage liver disease score was analyzed. The median terminal restriction fragment length of the hepatic tissues in biliary atresia was not significantly different from that of the control (p = 0.425), whereas the median normalized telomere-centromere ratio of hepatocytes in biliary atresia was significantly smaller than that of the control (p

J Oral Pathol Med (2012) 41: 372–378 Objectives: A precancerous condition is a lesion that, if left untreated, leads to cancer or can be induced to become malignant. In the oral region, leukoplakia is a lesion that has been regarded as precancerous. In cases of oral carcinoma, we have frequently noticed that a type of leukoplakia histologically demonstrating hyper-orthokeratosis and mild atypia (ortho-keratotic dysplasia; OKD) is often associated with carcinoma, either synchronously or metachronously. Therefore, we consider OKD-type leukoplakia to be a true precancerous lesion. Materials and Methods: In an attempt to clarify the relationship between OKD as a precancerous condition in the oral mucosa and telomere length, we estimated telomere lengths in this type of leukoplakia using quantitative fluorescence in situ hybridization, and also quantified the frequency of anaphase–telophase bridges (ATBs) in comparison with squamous cell carcinoma in situ (CIS) and the background tissues of CIS and OKD. Results: Ortho-keratotic dysplasia was frequently associated with squamous cell carcinoma (45.0%) and showed significantly shorter telomeres than normal control epithelium, CIS, or the background of CIS or OKD. The frequency of ATBs was much higher in OKD than in control epithelium or CIS. Conclusion: Ortho-keratotic dysplasia appears to be frequently associated with carcinoma, chromosomal instability, and excessively shortened telomeres, not only in the lesion itself but also in the surrounding background. Therefore, when this type of leukoplakia is recognized in the oral region, strict follow-up for oral squamous cell carcinoma is necessary, focusing not only on the areas of leukoplakia, but also the surrounding background.

Telomeres are repetitive G-rich DNA sequences located at the ends of chromosomes. Chromosomal and genomic instability due to telomere dysfunction plays an important role in carcinogenesis. To study telomere shortening in the oesophageal epithelium of alcoholics, we measured the telomere lengths of basal and parabasal cells in comparison with those of non-alcoholics using Q-FISH and our original software, Tissue Telo, and also assessed histological inflammation. Telomeres in basal cells were significantly shorter in alcoholics than in age-matched normal controls. Prominent histological findings of chronic inflammation were not evident in either alcoholics or non-alcoholics. Our finding that telomeres in the oesophageal epithelium are shorter in alcoholics than in non-alcoholics indicates that telomere shortening may be associated with the frequent occurrence of squamous cell carcinoma in alcoholics. Further studies to clarify the reason for the large annual loss of telomere length with rapid turnover or lower telomerase activity in the oesophageal epithelium of alcoholics will be necessary.

We monitored the telomere lengths and chromosomal instability characteristics of fibroblasts at different population doubling levels (PDLs) to gain further insight into the role of telomere shortening in chromosomal instability. We used 7 normal diploid human fibroblast strains (TIG-1, 3, 7, 103, 104, 112, and 114) and a quantitative fluorescence in situ hybridization method to measure telomere lengths of the p- and q-arms of individual chromosomes. We also enumerated morphologic signs of chromosomal instability, including fusion or loss of chromosomes, and anaphase bridges. In strains TIG-1, 3, 7, 103, and 114 at the late (phase 3) stage (≧40PDLs), 29 (96.6%) of 30 fusions were associated with one or both of the chromosomal arms that bear significantly shorter telomeres in those populations. In TIG-1 at 62PDL, 6 fusions were associated with Xq (n=3), 21q (n=3), and other (n=6) chromosomes. Xq and 21q had significantly shorter telomeres, and anaphase bridges were often associated with chromosomes X and/or 21 (74.6%). Our results indicate that chromosomes having excessively shortened telomeres at late PDLs begin to show features of instability such as fusions and anaphase bridges.

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere dysfunction in the surrounding background epithelium of squamous cell carcinoma in situ (CIS) of the oesophagus, we measured telomere lengths of basal and parabasal cells of epithelia with and without CIS using quantitative fluorescence in situ hybridization (Q-FISH) and our original software, Tissue Telo. Additionally, we assessed histological inflammation and the anaphase bridge index. In non-cancerous epithelium, telomeres in basal cells were significantly longer than those in parabasal cells, whereas CIS showed a homogeneous telomere pattern in the basal and parabasal cells. Telomeres in basal and parabasal cells were significantly shorter in the background with CIS than in epithelium from age-matched normal controls. Significant negative correlation was observed between the normalized telomere : centromere ratio (reflected telomere length) and the anaphase bridge index in non-cancerous epithelia from both normal controls and the CIS background with no histological inflammation. These findings indicate that tissue stem cells may be located among basal cells, and that telomere length distribution in component cell types differs between CIS and non-cancerous epithelium. We have demonstrated conclusively that oesophageal CIS arises from epithelium with short telomeres and chromosomal instability in the absence of histological inflammation.

Aging appears to be intrinsically related to carcinogenesis. Genomic instability due to telomere shortening plays an important role in carcinoma development. In order to clarify telomere dysfunction in carcinoma development, we examined the uninvolved epithelium adjacent to carcinoma in situ (CIS), i.e. background of CIS, and CIS itself, compared to control without carcinoma, using an improved quantitative fluorescence in situ hybridization (Q-FISH) method. We also estimated anaphase bridge (AB), which is inferred to be related to chromosomal instability. In all cell types (basal, parabasal, and suprabasal), mean telomere lengths were significantly shorter in the background than in the control. We also demonstrated increased incidences of AB, not only in CIS, but also in the background and control epithelia with excessively shortened telomeres. Thus we have conclusively demonstrated that CIS arises from epithelium with short telomeres.

We investigated the telomere lengths of individual cell types in lingual mucosa using an improved tissue quantitative fluorescence in situ hybridization (Q-FISH) method. Our tissue Q-FISH method compensates for partially cut nuclei in a tissue section by using the telomere:centromere ratio (TCR). We normalized our TCR measurements (NTCR) using a section from a block of cultured cells placed on the same slide, thus improving the accuracy and reproducibility of the results. Normal lingual mucosa was obtained from 21 autopsied individuals. Immunohistochemistry showed positivity mainly for p27, p63, and CK19 in basal cells, and for Ki-67 in parabasal cells. Q-FISH revealed that NTCR was significantly highest in basal cells and lowest in prickle cells, and also that telomere length regressed at a certain rate in each cell type, firstly. Significant correlations of NTCR among the three epithelial cell types were demonstrated. The present findings appear to support the theory that stem cells exist in the basal layer of the lingual epithelium. The reduction of telomere length with age and in each cell layer is consistent with the telomere biology theory of cell proliferation and differentiation in oral mucosa.

Little is known about the genes that regulate telomere length diversity between mammalian species. A candidate gene locus was previously mapped to a region on distal mouse Chr 2q. Within this region, we identified a gene similar to the dog-1 DNA helicase-like gene in C. elegans. We cloned this Regulator of telomere length (Rtel) gene and inactivated its expression in mice. Rtel(-/-) mice died between days 10 and 11.5 of gestation with defects in the nervous system, heart, vasculature, and extraembryonic tissues. Rtel(-/-) embryonic stem cells showed telomere loss and displayed many chromosome breaks and fusions upon differentiation in vitro. Crosses of Rtel(+/-) mice with Mus spretus showed that Rtel from the Mus musculus parent is required for telomere elongation of M. spretus chromosomes in F1 cells. We conclude that Rtel is an essential gene that regulates telomere length and prevents genetic instability.

Background: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>105) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). Methods: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T2AG3)n sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. Results: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10–15%) can be obtained. Conclusions: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells. Cytometry 36:267–278, 1999. © 1999 Wiley-Liss, Inc.

Human ovarian surface epithelial (HOSE) cells transfected with the E6 and E7 oncogenes of the human papilloma virus (PV) do not express measurable telomerase activity. Relative to untransfected control cells, HOSE-PV cells have an extended in vitro lifespan characterized by a very high frequency of telomeric associations (TAs) of chromosomes. In order to study the role of telomere shortening in the formation of TAs, we studied the telomere length in 120 dicentric chromosomes in HOSE-PV cells by using quantitative fluorescence in situ hybridization. Forty percent of the dicentric chromosomes had no fluorescence signal at the junction site, and in the remainder the fluorescence at the junction was less than at corresponding unjoined ends. These observations support a critical role of telomere shortening in the development of TAs and the subsequent genetic instability observed in a majority of tumor cells.

Human chromosomes terminate in a series of T2AG3 repeats, which, together with associated proteins, are essential for chromosome stability. In somatic cells, these sequences are known to be gradually lost through successive cells divisions; however, information about changes on specific chromosomes is not available. Individual telomeres could mediate important biological effects as was shown in yeast, in which loss of a single telomere results in cell-cycle arrest and chromosome loss. We now demonstrate by quantitative fluorescence in situ hybridization (Q-FISH; ref. 7) that the number of T2AG3 repeats on specific chromosome arms is very similar in different tissues from the same donor and varies only to some extent between donors. In all sixteen individuals studied, telomeres on chromosome 17p were shorter than the median telomere length--a finding confirmed by analysis of terminal restriction fragments from sorted chromosomes. These observations provide evidence of chromosome-specific factors regulating the number of T2AG3 repeats in individual telomeres and raise the possibility that the relatively short telomeres on chromosome 17p contribute to the frequent loss of 17p alleles in human cancers.

Along with the increasing need for living-donor liver transplantation (LDLT), the issue of organ shortage has become a serious problem. Therefore, the use of organs from elderly donors has been increasing. While the short-term results of LDLT have greatly improved, problems affecting the long-term outcome of transplant patients remain unsolved. Furthermore, since contradictory data have been reported with regard to the relationship between donor age and LT/LDLT outcome, the question of whether the use of elderly donors influences the long-term outcome of a graft after LT/LDLT remains unsettled. To address whether hepatocyte telomere length reflects the outcome of LDLT, we analyzed the telomere lengths of hepatocytes in informative biopsy samples from 12 paired donors and recipients (grafts) of pediatric LDLT more than 5 years after adult-to-child LDLT because of primary biliary atresia, using quantitative fluorescence in situ hybridization (Q-FISH). The telomere lengths in the paired samples showed a robust relationship between the donor and grafted hepatocytes (r = 0.765, p = 0.0038), demonstrating the feasibility of our Q-FISH method for cell-specific evaluation. While 8 pairs showed no significant difference between the telomere lengths for the donor and the recipient, the other 4 pairs showed significantly shorter telomeres in the recipient than in the donor. Multiple regression analysis revealed that the donors in the latter group were older than those in the former (p = 0.001). Despite the small number of subjects, this pilot study indicates that donor age is a crucial factor affecting telomere length sustainability in hepatocytes after pediatric LDLT, and that the telomeres in grafted livers may be elongated somewhat longer when the grafts are immunologically well controlled.

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T 2 AG 3 ) n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T 2 AG 3 ) n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere shortening in the epidermis surrounding actinic keratosis, we measured telomere lengths of basal, parabasal, and suprabasal cells in epidermis with actinic keratosis (actinic keratosis group, n = 18) and without actinic keratosis (sun-protected, n = 15, and sun-exposed, n = 13 groups) and in actinic keratosis itself as well as in dermal fibroblasts in the 3 groups, using quantitative fluorescence in situ hybridization. Among the 3 cell types, telomeres of basal cells were not always the longest, suggesting that tissue stem cells are not necessarily located among basal cells. Telomeres of basal cells in the sun-exposed group were shorter than those in the sun-protected group. Telomeres in the background of actinic keratosis and in actinic keratosis itself and those of fibroblasts in actinic keratosis were significantly shorter than those in the controls. Our findings demonstrate that sun exposure induces telomere shortening and that actinic keratosis arises from epidermis with shorter telomeres despite the absence of any histologic atypia.

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage.

Platinum-based combination chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC). While cisplatin is effective, its use is not curative and resistance often emerges. As a consequence of microenvironmental heterogeneity, many tumour cells are exposed to sub-lethal doses of cisplatin. Further, genomic heterogeneity and unique tumor cell sub-populations with reduced sensitivities to cisplatin play a role in its effectiveness within a site of tumor growth. Being exposed to sub-lethal doses will induce changes in gene expression that contribute to the tumour cell's ability to survive and eventually contribute to the selective pressures leading to cisplatin resistance. Such changes in gene expression, therefore, may contribute to cytoprotective mechanisms. Here, we report on studies designed to uncover how tumour cells respond to sub-lethal doses of cisplatin. A microarray study revealed changes in gene expressions that occurred when A549 cells were exposed to a no-observed-effect level (NOEL) of cisplatin (e.g. the IC10). These data were integrated with results from a genome-wide siRNA screen looking for novel therapeutic targets that when inhibited transformed a NOEL of cisplatin into one that induced significant increases in lethality. Pathway analyses were performed to identify pathways that could be targeted to enhance cisplatin activity. We found that over 100 genes were differentially expressed when A549 cells were exposed to a NOEL of cisplatin. Pathways associated with apoptosis and DNA repair were activated. The siRNA screen revealed the importance of the hedgehog, cell cycle regulation, and insulin action pathways in A549 cell survival and response to cisplatin treatment. Results from both datasets suggest that RRM2B, CABYR, ALDH3A1, and FHL2 could be further explored as cisplatin-enhancing gene targets. Finally, pathways involved in repairing double-strand DNA breaks and INO80 chromatin remodeling were enriched in both datasets, warranting further research into combinations of cisplatin and therapeutics targeting these pathways.

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm9 mouse reference genome assembly contains at least 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments have persisted through several iterations of the mouse reference genome and have been difficult to detect using conventional sequencing techniques. The ability to map SCE events at high resolution and fine-tune reference genomes by Strand-seq dramatically expands the scope of single-cell sequencing.

Trisomies 18 and 21 are genetic disorders in which cells possess an extra copy of each of the relevant chromosomes. Individuals with these disorders who survive birth generally have a shortened life expectancy. As telomeres are known to play an important role in the maintenance of genomic integrity by protecting the chromosomal ends, we conducted a study to determine whether there are differences in telomere length at birth between individuals with trisomy and diploidy, and between trisomic chromosomes and normal chromosomes. We examined samples of peripheral blood lymphocytes (PBLs) from 31 live neonates (diploidy: 10, trisomy 18: 10, trisomy 21: 11) and estimated the telomere length of each chromosome arm using Q-FISH. We observed that the telomeres of trisomic chromosomes were neither shorter nor longer than the mean telomere length of chromosomes as a whole among subjects with trisomies 18 and 21 (intra-cell comparison), and we were unable to conclude that there were differences in telomere length between 18 trisomy and diploid subjects, or between 21 trisomy and diploid subjects (inter-individual comparison). Although it has been reported that telomeres are shorter in older individuals with trisomy 21 and show accelerated telomere shortening with age, our data suggest that patients with trisomies 18 and 21 may have comparably sized telomeres. Therefore, it would be advisable for them to avoid lifestyle habits and characteristics such as obesity, cigarette smoking, chronic stress, and alcohol intake, which lead to marked telomere shortening.

Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduced telomerase activity. Healthy individuals showed a broad range in average telomere length in granulocytes and lymphocytes at any given age. The average telomere length declined with age at a rate that differed between age-specific breakpoints and between cell types. Gender differences between leukocyte telomere lengths were observed for all cell subsets studied; interestingly, this trend could already be detected at birth. Heterozygous carriers for mutations in either the telomerase reverse transcriptase (hTERT) or the telomerase RNA template (hTERC) gene displayed striking and comparable telomere length deficits. Further, non-carrier relatives of such heterozygous individuals had somewhat shorter leukocyte telomere lengths than expected; this difference was most profound for granulocytes. Failure to maintain telomere homeostasis as a result of partial telomerase deficiency is thought to trigger cell senescence or cell death, eventually causing tissue failure syndromes. Our data are consistent with these statements and suggest that the likelihood of similar processes occurring in normal individuals increases with age. Our work highlights the essential role of telomerase in the hematopoietic system and supports the notion that telomerase levels in hematopoietic cells, while limiting and unable to prevent overall telomere shortening, are nevertheless crucial to maintain telomere homeostasis with age., Author Summary Human blood cells all originate from a common precursor, the hematopoietic stem cell. Telomerase, the enzyme responsible for adding telomere repeats to chromosome ends, is active in human hematopoietic stem cells but appears unable to maintain a constant telomere length with age. We first document the telomere length of different blood cell subsets from 835 healthy individuals between birth and 100 years, to delineate the normal rate of telomere attrition with age. Telomere lengths of blood cells were found to be slightly longer in women than in men, from birth and throughout life. We then compared this reference data to the telomere length in similar blood cell subsets from individuals with reduced telomerase activity as a result of a mutation in one of the genes encoding telomerase and from their direct relatives. Strikingly short telomeres were found in telomerase-deficient individuals, consistent with their cellular pathology and disease susceptibility, and somewhat shorter telomeres than expected were found in cells of relatives with normal telomerase maintenance. Our data can be used as a reference for blood cell telomere length in studies of normal and accelerated aging.

Different methods are investigated in selecting and generating the appropriate microscope images for analysis of three-dimensional objects in quantitative microscopy. Traditionally, the ‘best’ focused image from a set is used for quantitative analysis. Such an objectively determined image is optimal for the extraction of some features, but may not be the best image for the extraction of all features. Various methods using multiple images are here developed to obtain a tighter distribution for all features. Three different approaches for analysis of images of stained cervical cells were analyzed. In the first approach, features are extracted from each image in the set. The feature values are then averaged to give the final result. In the second approach, a set of varying focused images are reconstructed to obtain a set of in-focus images. Features are then extracted from this set and averaged. In the third approach, a set of images in the three-dimensional scene is compressed into a single two-dimensional image. Four different compression methods are used. Features are then extracted from the resulting two-dimensional image. The third approach is employed on both the raw and transformed images. Each approach has its advantages and disadvantages. The first approach is fast and produces reasonable results. The second approach is more computationally expensive but produces the best results. The last approach overcomes the memory storage problem of the first two approaches since the set of images is compressed into one. The method of compression using the highest gradient pixel produces better results overall than other data reduction techniques and produces results comparable to the first approach.

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast1 and sister chromatid segregation is not random in Escherichia coli2. Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms3. This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental “Watson” and “Crick” DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (Chromosome Orientation Fluorescence in situ Hybridization4) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to exhibit a uniform orientation with the 5′ end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant (p

High-content microscopic screening systems are powerful tools for extracting quantitative multiparameter measures from large number of cells under numerous conditions. These systems perform well in applications that monitor the presence of objects, but lack in their ability to accurately estimate object intensities and summarize these findings due to variations in background, aberrations in illumination, and variability in staining over the image and/or sample wells. We present effective and automated methods that are applicable to analyzing intensity-based cell cycle assays under high-throughput screening conditions. We characterize the system aberration response from images of calibration beads and then enhance the detection and segmentation accuracy of traditional algorithms by preprocessing images for local background variations. We also provide a rapid, adaptive, cell-cycle partitioning algorithm to characterize each sample well based on the estimated locally and globally corrected cell intensity measures of BrdU and DAPI incorporation. We demonstrated the utility and range of our cell ploidy and probe density measurement methods in a pilot screen using a siRNA library against 779 human protein kinases. With our method, multiple image-based quantitative phenotypes can be realized from a single high-throughput image-based microtiter-plate screen.

Critically shortened, dysfunctional telomeres may play a role in the genetic instabilities commonly found in cancer. We analyzed 30 surgical specimens of invasive breast carcinoma from women aged 34 to 91 years and estimated telomere lengths as telomere-to-centromere ratio values in the 5 different cell types comprising breast tissue in order to clarify telomere length variations within and between individuals using our tissue quantitative fluorescence in situ hybridization method. We obtained 3 novel findings. (1) In corresponding normal tissues, telomere length decreased in the order myoepithelial cells > normal-appearing fibroblasts > luminal epithelial cells, and telomere lengths were characteristic in these 3 cell types within each individual. (2) As expected, cancer cells had significantly shorter telomeres than myoepithelial cells (P < .0001) and normal-appearing fibroblasts (P = .0161), but there was no significant difference in telomere length between luminal cells and cancer cells (P = .6270). (3) Fibroblasts adjacent to cancer had longer telomeres than normal-appearing fibroblasts distant from cancer (P < .0001). This study, which represents the first reported assessment of telomere length variations in the 5 cell types comprising breast tissue within and between individuals, revealed that normal luminal epithelial cells and cancer cells had the shortest telomeres. Our new findings indicate that telomeres of background luminal cells are as short as those of cancer cells. Tissue quantitative fluorescence in situ hybridization, applicable to analysis of individual cells in tissue sections, is considered to be a powerful technique with considerable promise for studies in oncology.

The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 microns below to 25 microns above the optimal focus position. These limits were asymmetrical about ideal focus due to a pronounced asymmetry in the effects of positive and negative defocusing on the cell signal.

Critically shortened telomeres make chromosomes susceptible to the instability and widespread cytogenetic alterations that characterize most human cancers. We hypothesized that the very rapid cell proliferation observed in esophageal squamous cell carcinomas might accelerate telomere shortening and chromosomal instability associated with carcinogenesis. We used a number of telomere measurement techniques including quantitative fluorescence in situ hybridization (Q-FISH) to compare chromosomal aberrations and telomere lengths of individual chromosomes in esophageal squamous cell carcinomas (ESCCs) and nearby non-neoplastic esophageal epithelium (NNEE) cells. Our results showed that the mean telomere length in ESCC cells was significantly less than that in adjacent NNEE cells, and that telomeres in all NNEE cells were significantly shorter than those in normal esophageal epithelium (reported previously). In addition, there was no evidence linking telomere shortening of a particular chromosome to field cancerization in ESCC. However, a mechanistic link between telomere shortening and chromosomal instability was supported by a higher frequency of anaphase/telophase bridges and an increase in the frequency of aneuploidy. This study furthers our understanding of the mechanism by which telomere shortening and chromosomal instability lead to carcinogenesis and field cancerization in the esophagus.

The limited life span of most blood cells requires the continuous production of cells, which in adults exceeds 10(12) cells/day. This impressive production of cells (approximately 4 x 10(16) cells over a lifetime) is achieved by the proliferation and differentiation of committed progenitor cells, which themselves are derived from a population of pluripotent stem cells with self-renewal potential. Paradoxically, the large majority of stem cells in adult bone marrow are quiescent cells. One possibility is that stem cells, like other somatic cells, have only a limited replicative potential (< 100 divisions). This hypothesis is supported by two key observations and the consideration that, in theory, 55 divisions can yield 4 x 10(16) cells. First, it was shown that 'candidate' stem cells purified from fetal and adult tissue showed dramatic functional differences in turn-over time and the ability to produce cells with stem cell properties, Second, these functional differences were found to correlate with a measurable loss of telomere repeats despite the presence of low but readily detectable levels of telomerase in all purified cell fractions. In order to address questions about the role of telomeres in normal and malignant haemopoiesis, we developed a quantitative fluorescence in situ hybridization technique. Here we review the characteristics of this novel tool to assess the number of telomere repeats at the end of individual chromosomes and provide an overview of recent observations.

Objective: We have developed a novel method for evaluating telomere length in four different cell types in non-cancerous and cancerous mucosal tissue from 15 cases of squamous cell carcinoma of the esophagus using tissue quantitative fluorescence in situ hybridization (Q-FISH). We hypothesized that the very rapid cell proliferation observed in esophageal squamous cell carcinomas might accelerate the telomere shortening and chromosomal instability associated with carcinogenesis. Methods: Tissue Q-FISH and the telomere to centromere intensity ratio (TCR) were used to compare telomere shortening in tissue sections taken from esophageal squamous cell carcinomas and adjacent non-cancerous esophageal tissues. Results: The peak percentage of TCR was 1 for the non-cancerous esophageal cell types. Basal layer cells had the longest telomeres in comparison with prickle, cancer, and stromal cells, and strongly expressed hTERT, cytokeratin 14 and CD49f, but not MIB-1. Conclusion: These results suggest the presence of stem cells in the basal layer of the esophagus. Esophageal squamous cell carcinomas also display anaphase bridges, evidencing chromosomal instability.In conclusion, our TCR method can be used to distinguish between benign and malignant tissue in esophageal lesions. In order to apply this approach clinically to individual cases, further studies are in progress.

We developed a novel method for evaluating telomere length in 6 cell types of noncancerous and cancerous mucosal tissues from 11 cases of gastric neoplasm using the quantitative fluorescence in situ hybridization method with telomere and centromere peptide nucleic acid probes. Our telomere length estimates were determined from the background-corrected telomere intensity divided by the background-corrected centromere intensity (telomere-to-centromere ratio). Our results indicated that telomere lengths in each of the cases studied were reduced in turn from fibroblasts to fundic gland cells, to glandular neck cells, and then to surface foveolar cells. However, the telomere lengths of intestinalized cells located among fundic glands were not always shorter than those of the other cell types, as reported previously by others. Helicobacter pylori infection was suggested to induce the telomere shortening seen in the fundic glands. Although the mean telomere lengths varied among the 8 gastric cancer cases, correlation of the telomere lengths with the Ki-67 labeling index was established after normalization with the fibroblast measurements. We conclude that our telomere-to-centromere ratio method can reliably estimate the telomere lengths of the 6 cell types in the gastric mucosa and clarifies the relationship between proliferative activity and the telomere length of cancer cells.

One of the most aggressive human malignancies, anaplastic thyroid carcinoma (ATC), has an extremely poor prognosis that may be explained by its genomic instability. We hypothesized that the very rapid cell turnover observed in ATC might accelerate telomere shortening and chromosomal instability associated with tumor cell malignancy. To compare and measure chromosomal aberrations and telomere shortening in the anaplastic thyroid cancer cell line OCUT-1, we applied quantitative fluorescence in situ hybridization (Q-FISH) techniques. In all 15 metaphases studied, telomere length estimates from Q-FISH of chromosomes in ATC were shorter than those of a fibroblast cell line derived from the stroma adjacent to the carcinoma. OCUT-1 cells display several chromosomal abnormalities, but have a near-normal chromosome complement of 46, XX, making it easy to analyze the karyotype. The karyotype showed 50, XX, +7, +11, der(ll)t(3;ll)(q23;q23)x2, del(12)(pl 1.2p12), +20, +1mar. We analyzed carefully the abnormalities in karyotype of OCUT-1 associated with telomere shortening on each chromosome and expression of subtelomeres. Telomere lengths in the q-arms of the abnormal chromosome del(12)(p11.2p12) were shorter than the average length in the q-arms of the normal chromosome 12 in OCUT-1. Subtelomeres on the abnormal chromosome der(11)t(3;11)(q23;q23)x2 also showed loss of signals on 11p, but there was no loss of signals in the cytogenetically normal trisomies 7 and 20 or the abnormal chromosome del(12)(p11.2p12). Subtelomeres of 3q had eight signals, one pair remaining in place on 3q and another pair on the abnormal 11p. Our findings suggest that telomere shortening and subtelomere loss are correlated with genetic instability in this anaplastic thyroid carcinoma cell line.

The cell analyzer, an automated apparatus for biological studies of live cells, is discussed. The major enhancement of the cell signal is performed optically using a microscope in bright field microscopy mode. By appropriate selection and arrangement of the optics, a characteristic cell signal that can be easily distinguished from most debris signals is obtained. Several focusing algorithms are discussed, and the results of some recognition experiments are presented. >

This unit describes a quantitative technique for measuring the lengths of telomere repeat sequences in individual chromosomes from single metaphase cells. The technique is based on fluorescence in situ hybridization (FISH) adapted for use with peptide nucleic acid (PNA) probes. PNA is an example of novel synthetic oligonucleotide "mimetic" which has a higher affinity than regular oligonucleotide (RNA or DNA) probes for complementary single-strand (ss) DNA sequences. PNA oligonucleotides have excellent penetration properties due to their small size (typically 15 to 18-mers) and can be directly labeled with fluorochromes. These properties have been exploited to develop quantitative fluorescence in situ hybridization (Q-FISH) onto denatured single-stranded chromosomal DNA target sequences. The latter can be present in preparations of fixed metaphase cells on slides (Q-FISH) or in heat-treated (interphase) cells in suspension (flow-FISH).

Publisher Summary The physical ends of chromosomes or telomeres are in most species composed of G-rich repeat sequences and associated proteins. The DNA of telomeres consists of specific repetitive sequences (TTAGGG in all vertebrates) that are synthesized from an RNA template by a specialized reverse transcriptase called telomerase. Conventionally, Southern analysis is used to estimate the average length of telomere repeat sequences. For this purpose, DNA extracted from a population of cells is digested with restriction enzymes and analyzed by gel electrophoresis. With this method DNA fragments are separated on the basis of size. The telomere repeat sequences present in a minor fraction of the DNA fragments are visualized after hybridization with a radiolabeled probe specific for telomeric DNA. Information on the length of telomere repeats in individual chromosomes can also be extracted from digital images of metaphase chromosomes that are subjected to quantitative fluorescence in situ hybridization (Q-FISH) with directly labeled peptide nucleic acid (PNA) telomere probes.

The DNA of human chromosomes terminates in several kilobases of telomere repeats that are gradually lost with age and with replication in vitro. Defective telomere maintenance has been shown to be causally linked to cell cycle exit and apoptosis. In order to overcome the limitations imposed by Southern blotting, we have established a quantitative fluorescence in situ hybridization (Q-FISH) technique. This technique allows estimation of telomere length in specific chromosome arms from metaphase cell preparations. Furthermore, we have extended quantitative in situ hybridization to flow cytometry (flow FISH) in order to obtain information on the mean telomere repeat content in suspended cells. Telomere length in granulocytes, monocytes, CD8 and CD4 T lymphocytes and natural killer cells was found to differ slightly in the peripheral blood of adults. However, strikingly longer telomeres were observed in B lymphocytes (∼ 1.3 kb longer), suggesting a functional role for telomere maintenance in this cell subset. In summary, Q-FISH and flow FISH represent new methods for measuring telomere length in single cells and allow studies of telomere dynamics in haematopoietic subpopulations at various stages of normal and abnormal antigen responses.

The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1–2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5–1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence.

Aim Process automation is an important requirement for streamlining and standardizing technical procedures, such as cell isolation, in a routine laboratory. We have previously developed RoboSep™, the first instrument to fully automate the cell labeling and separation steps of an immunomagnetic cell isolation procedure. RoboSep™ uses EasySep™ reagents and protocols and can isolate up to 4 individual samples at one time. More recently we have developed a high throughput version of RoboSep™, called RoboSep-HT, which can isolate 16 samples from up to 4 different cell types in about 1 hour. Like RoboSep™, RoboSep-HT uses disposable filter tips to avoid cross contamination between samples and is operated using an intuitive graphical user interface. Sample barcoding is a standard feature. In this study, positive selection of CD3+ and myeloid+ cells directly from whole blood using EasySep™ reagents was performed on the new RoboSep-HT and compared to performance using the classic RoboSep™. Methods 1mL of fresh, 24-hour or 48-hour old whole blood was diluted with an equal volume of 1xRBC lysis buffer and loaded on each instrument. Purity (flow cytometry), recovery (cell counts) and DNA yield (OD) were assessed following isolation using each robot. Replicates were averaged prior to statistical analysis (t-tests). Results There was no significant difference in either purities, cell recoveries or DNA yields when CD3 and myeloid cell isolation was performed on the RoboSep-HT versus the classic RoboSep™. [ Table 1 ] Conclusions RoboSep-HT can be used for the high throughput isolation of multiple cell types and is ideally suited for cell isolation prior to chimerism testing. McQueen: STEMCELL Technologies: Employee. Kellerman: STEMCELL Technologies: Employee. Frehlick: STEMCELL Technologies: Employee. Gromadzki: STEMCELL Technologies: Employee. Poon: STEMCELL Technologies: Employee. Woodside: STEMCELL Technologies: Employee. Thomas: STEMCELL Technologies: Employee.

Chromoendoscopy with Lugol iodine staining provides important information on the development of squamous cell carcinoma (SCC). In particular, distinct iodine-unstained lesions (DIULs) larger than 10 mm show a high prevalence in high-grade intraepithelial neoplasia. It has also been reported that inactive ALDH2*1/*2 and less-active ADH1B*1/*1, and smoking, are risk factors for esophageal SCC. We previously examined telomere shortening in the esophageal epithelium of alcoholics, and suggested a high prevalence of chromosomal instability in such individuals. In the present study, we attempted to analyze telomere lengths in 52 DIULs with reference to both their size and multiplicity, ALDH2 and ADH1B genotypes, and smoking history. Patients with DIULs

A digital camera has been designed based on a 1317 by 1035 pixel, full frame, CCD which offers the large area, high density sampling but with the options of programmable sub-area scanning and multiple independent x and y binning and programmable gain and offset. The sub-area scanning and binning can be used to increase the effective frame rate of the device for such applications as fast focusing and object location. The binning also provides greater sensitivity at the cost of spatial resolution, but is ideal in low light level applications such as fluorescence microscopy. The programmability of this camera permits the switching between the various clocking modes within a single frame time. Hence, the image can be auto-focused in sub-area scanning mode and then within a single frame time the full frame image can be acquired. The programmable clocking modes make this device ideal for quantitative imaging applications requiring a high throughput and the programmable gain and offset allow the user to fine tune the device for his specific needs. A description of the programmable microimager design and some initial measurements of noise, linearity, frequency response, and stability are presented.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Objective The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation, expansion, and differentiation. Materials and Methods We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives), together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses, Affymetrix profiling, real-time PCR, and immunoprecipitation. Results Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g., POU5F1 , NANOG , SOX2 , FOXD3 ). siRNA analyses identified two retinoblastoma binding proteins (RBBP4, RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells, and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1 , NANOG , SOX2 , and/or FOXD3 plus certain cell cycle genes (e.g., CCNA2 , CCNB1 ), while increasing expression of genes involved in organogenesis (particularly neurogenesis). Conclusions These results reveal new candidate positive regulators of human PS cells, providing evidence of their ability to regulate expression of pluripotency, cell cycle, and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency.

Background Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. Results We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. Conclusions Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.

To reconstruct the object from its observed images, the characteristics of the imaging system must first be obtained. In a microscope imaging system, the characteristics vary not only in the imaging plane, but also vary as a function of the focus in which the image is taken. Thus, a three dimensional system response or point spread function (PSF) needs to be determined. One way of determining the PSF is to use a theoretical approach to analyze the aberration-free microscope imaging system. However, the assumptions and properties of lenses in the system are often not ideal. Thus an experimental approach for determining the PSF is sometimes used. We report on the results of our experiments where some of the problems associated with the determination of the experimental PSF are overcome. Point source objects are hard to find in nature. In our analysis, we use test objects which simulates point sources, (such as small fluorescence beads,) and objects which can be described as a convolution of the PSF with their shape (such as a step edge). To increase the spatial resolution, the precise location of the object is also estimated to a fraction of a pixel. The results of this is then compared with the theoretical approach.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

A new generation, large frame (1317 X 1035 pel) digital camera, based on the MicroImager 1400 (MI1400), has been developed. The new MicroImager 1400-12 (MI1400- 12) uses improved noise reduction circuits to allow for 12 bit digitization up to 8 Mpel/s. Methods of correlated double sampling were investigated and a clamp-and-sample design was chosen. The MicroImager is a full frame device with a 100% fill factor. Integration time, readout frequency and pixel additive mode (binning) are under computer control. The camera outputs digital, RS-422 compatible data to the host frame memory through a 50-pin high density connector. The system is compatible with various (E)ISA and VME based imaging boards. The applications are quantitative digital microscopy in brightfield and fluorescence and in numerous industrial and medical camera applications. A number of comparative performance measurements were defined and carried out including spatial resolution (impulse response), photometric response (linearity), sensitivity, dynamic range, signal-to-noise ratio and other noise parameters. The MI1400-12 has a slightly improved impulse response. When used with primary image plane optics, the MicroImager shows a 6% improvement in the spatial resolution over standard C-mount compensation optics. In further comparisons the linearity of the MI1400-12's response holds to a coefficient of regression larger than 0.999 and has a four-fold increase in sensitivity over the MI1400. A dynamic range of 61 dB was measured for single frames corresponding to an 8 dB increase. For a 30 frame average, 77 dB was achieved. Circuit noise parameters (random noise at dark levels) improved by a factor of 3 to 4.

In bright field microscopy, quantitative analysis of acquired images is customarily performed using the `best' image. Since an image with sufficient detail and clarity is required for consistent classification and discrimination of objects in the image, the image with higher magnification is commonly chosen for the analysis, which generally corresponds to lower focal depths. The objectively determined `best' focus level, although optimal for the extraction of some features from the chosen objects, may not correspond to the best focal level for the extraction of some other features. To obtain tighter distribution of all features, we have been searching for a method which employs analysis of images acquired at different focal planes. In this work, we analyzed images of stained cervical cells using three different approaches. In the first approach, different features were extracted from images taken at different focal planes. In the second approach, we used simultaneously all the in-focus and out-of-focus information from the images to reconstruct the focussed images at various focal planes. In the third approach, the in-focus three-dimensional scene was compressed to two dimensions to simulate an image taken from a system with a very large depth of focus. The latter method reduced the data storage size and simplified subsequent scene analysis. The advantages and disadvantages of the above approaches are discussed.© (1992) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.