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PubMed1999

Comparison of several digital and stereological methods for estimating surface area and volume of cells studied by confocal microscopy.

BACKGROUND: The implementation of different methods for estimating the surface area and volume of cells studied by confocal microscopy was developed. The methods were compared from the point of view of their precision, applicability and efficiency. METHODS: Interactive stereological methods (spatial grid method, fakir method, Cavalieri principle) as well as automatic digital methods (digital Crofton method, voxel counting, triangulation method, iso-intensity contouring method) were considered. The methods were tested on model geometrical solids and on real volume images consisting of a stack of serial sections encompassing entire tobacco BY-2 cells or cell chains. RESULTS: It is shown that many of the studied methods are very precise when applied to cells of simple or moderately complex shapes. The automatic digital methods are fast and precise but their applicability is limited by the necessity to segment automatically the object surface and to find an optimal resolution. This limitation is not present in stereological methods which are applied interactively and thus are more time-consuming. CONCLUSIONS: The presented implementations of the fakir method and the Cavalieri principle enable interactive, unbiased and efficient estimation of the cell surface area and volume. The recommended steps for measuring the surface area and/or volume of objects studied by confocal microscopy are described.

Cell Line↗
PubMed1999

Quantitative assessment of the alteration of chromatin during the course of FISH procedures. Fluorescent in situ hybridization.

BACKGROUND: DNA denaturation, required for fluorescent in situ hybridization (FISH) experiments, is likely to induce chromatin alterations. Only few attempts have been made to quantify the extent of these perturbations. We propose a quality-control approach based on image analysis to monitor the effect of a procedure commonly used in FISH experiments. METHODS: Using DAPI as a probe, the same nuclei were successively imaged with a CCD camera after fixation, after permeabilization, and after thermal denaturation and hybridization with a centromeric probe. The modifications of the staining pattern were analyzed. Volumes of the FISH signals were measured using confocal imaging. RESULTS: DAPI staining combined with image analysis proved to be a sensitive tool to visualize the effects of different treatments used in FISH experiments. Permeabilization of nuclei after fixation has only limited impact on the chromatin. On the contrary, the denaturation procedure modifies the staining of DNA by DAPI, as well as the underlying chromatin structure as assessed by the increase of FISH signal volume with denaturation time. The protocol that involves a pre-fixation permeabilization step results in a more severe loss of chromatin structure. CONCLUSIONS: Our results clearly show that analysis of alterations of DAPI staining patterns is a useful monitoring tool to control and standardize hybridization procedures.

Amnion↗
PubMed1999

Nuclear morphometry as an intermediate endpoint biomarker in chemoprevention of cervical carcinoma using alpha-difluoromethylornithine.

The use of nuclear morphometry as an intermediate endpoint biomarker is described in a Phase I, dose-seeking trial of chemoprevention of cervical cancer, using the agent alpha-difluoromethylornithine (DFMO). Thirty patients with grade III cervical intraepithelial neoplasia (CIN III) were enrolled, and these received daily doses of DFMO at 0.06-1.0 mg/m(2) for a period of 1 month. Fifteen patients were observed to have a complete or partial regressive response to the agent, as assessed by histopathology. No significant differences in cell feature measurements were found between responders and nonresponders in specimens obtained before treatment, indicating that it may be difficult to predict response on the basis of these measurements. In specimens collected after treatment, large differences in morphometric features were observed between responders and nonresponders, indicating a differential effect of DFMO. Significantly modulated features were considered in terms of their correlations with CIN grade, which was determined from an independent set of measurements from archival tissue. Differences between features were consistent with a deletion of cells with high grade nuclei in the responders, and with the persistence of a more heterogeneous population of high grade cells in the nonresponders. Based on an independent set of measurements from archival material, a morphometric index of progression was derived, yielding a quantitative measure of the degree of nuclear atypia in these lesions. When applied to this trial, the morphometric index was seen to be specifically and consistently decreased in responsive lesions, and unchanged in nonresponders. The study indicates that morphometric features fulfill the requirements for an intermediate endpoint biomarker of cervical cancer chemoprevention.

Antineoplastic Agents↗
PubMed1999

AutoPap system detection of infections and benign cellular changes: results from primary screener clinical trials.

Primary screening devices for cervical cytology must show performance data for the detection of infectious organisms and benign cellular changes (BCC) for cytologists who routinely report these findings. The data on infection and BCC from the AutoPap primary screening clinical trials are presented herein. The presence of infectious organisms (candida, trichomonas, shift in bacterial flora, herpes, actinomyces) and BCC were noted in each of the clinical trial arms (current practice, CP; AutoPap-assisted practice, AP). For the purposes of these analyses, a report of infection or BCC from either arm was considered to be "truth." In 25,124 slides analyzed, there were 2,925 cases of infection identified. Of these, CP identified 2,141, and AP identified 1,985. The overall detection results are statistically equivalent. Of 17 cases of actinomyces, CP detected 8, while AP detected 12. Of 1,282 cases of candida, CP detected 983, and AP detected 865. Of 1,375 cases of shift of bacterial flora, CP detected 897, and AP detected 869. Of 14 cases of herpes, CP detected 9, and AP detected 11. Of 343 cases of trichomonas, CP detected 293, and AP detected 275. There were 5,156 cases of BCC identified in the trial. CP detected 3,431, and AP detected 3,276. The detection rates for BCC are statistically equivalent. The results show that the AutoPap-assisted practice for the primary screening of conventional cervical cytology slides is equivalent to the current practice for the detection of cervical infections and benign cellular changes.

Actinomyces↗
PubMedDate not supplied

Nuclear DNA content and p53 immunostaining in metachronous preneoplastic lesions and subsequent carcinomas of the oral cavity.

BACKGROUND: Clinical evaluation of preneoplastic lesions of the oral cavity is difficult. Histopathologic grading of dysplasias shows large variability and does not give reliable information concerning the risk for progression to cancer. METHODS: DNA image cytometry and p53 immunostaining were performed to describe the pattern of DNA aberration and p53 overexpression in confined preneoplastic lesions and in the subsequent carcinomas developing at the same site in 20 patients. RESULTS: Hyperplastic and/or inflammatory lesions showed a diploid DNA pattern in 81% of the cases and 23% were p53-positive. Dysplastic preneoplastic lesions showed a nondiploid/ aneuploid DNA pattern in 73% and 64% were p53-positive. The subsequent invasive carcinomas were nondiploid/aneuploid in 86% and p53-positive in 69% of cases. CONCLUSIONS: Analysis of nuclear DNA content and p53 immunostaining appears to be useful as an adjunct to histopathology in the evaluation of true precancerous lesions.

Adult↗
PubMed2000

Effect of ethanol on muscarinic receptor-induced calcium responses in astroglia.

The effects of ethanol on muscarinic receptor-mediated calcium responses were investigated in individual primary rat astrocytes and human 132 1N1 astrocytoma cells using indo-1/AM and image cytometry. After a 30-min incubation, carbachol-induced calcium responses were inhibited only at 100 or 250 mM ethanol. The effects of ethanol were more pronounced and occurred at lower concentrations with longer exposures, with significant inhibition seen at 10 mM following a 24-hr incubation. Thapsigargin- and glutamate-induced responses were unaffected by ethanol, indicating some selectivity in this inhibition. Upon removal of ethanol, inhibition of calcium responses persisted for up to 6-12 hr, with carbachol responses returning to control levels by 24 hr after washout. Ethanol exposure did not affect muscarinic-receptor binding in astrocytoma cells, but inhibited carbachol-induced IP(3) formation. Inhibition of (3)H-thymidine incorporation by ethanol also persisted upon removal of the alcohol, with a time-dependency similar to that of the calcium responses. These results indicate that ethanol inhibits muscarinic receptor-induced calcium responses in astroglia in a concentration- and duration-dependent manner. They also show that co-incubation with ethanol is not necessary for this effect, suggesting that long-term exposure to ethanol may modify, in a reversible manner, the coupling of muscarinic receptors with its effector. This effect of ethanol may play a role in ethanol's inhibition of carbachol-induced thymidine incorporation.

Animals↗
PubMed1999

In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics.

The biocompatibility of two implantable materials, zirconia and alumina ceramics, was investigated in vitro using human osteoblast cell cultures. The viability of osteoblast cells with the materials was evaluated by the methylthiazole sulfate test that revealed an absence of any cytostatic or cytotoxic effect. Cell proliferation kinetic and total protein synthesis in osteoblasts with zirconia or alumina were similar to that observed in control cells cultured on glass coverslips. Light and scanning electron microscopic examinations showed an intimate contact between osteoblasts and the substrates; well-spread cells were observed on the surfaces of both materials. Adhesion ability and morphological characteristics were preserved in osteoblast cultures with these substrates. Moreover, immunohistochemical staining in osteoblasts with zirconia and alumina showed the capacity of these cells to elaborate the extracellular matrix composed of types I and V collagen, osteocalcin, osteonectin, bone sialoprotein, and cellular fibronectin. Finally, DNA image cytometry and interphase silver-nucleolar organizer regions quantification were applied as complementary biocompatibility tests to detect any changes in DNA synthesis and cell proliferation, respectively. The results showed that neither material altered cell ploidy or cell growth rate in accordance with the absence of any inducing effect on DNA synthesis or proliferation.

Aluminum Oxide↗
PubMed1993

Prognostic value of DNA analysis in colorectal carcinoma.

BACKGROUND: Reported experiences regarding the prognostic significance of DNA content in colorectal carcinoma have been a matter of controversy. METHODS: DNA analysis with image cytometry was performed in 137 patients with colorectal cancer. Only patients who had resection without tumor residual and who did not die postoperatively as a consequence of the operation were entered in the study. At the time of DNA analysis, neither the histomorphologic data nor the relapse-free survival time of the patients were known. RESULTS: In this investigation the DNA content of tumor cells had no univariate or multivariate influence on the relapse-free survival time. The prognosis was dependent on the tumor localization, depth of tumor infiltration, lymph node metastasis, and grade. CONCLUSIONS: DNA content provides no additional prognostic information in colorectal carcinoma.

Adult↗
PubMed2001

Automatic signal classification in fluorescence in situ hybridization images.

BACKGROUND: Previous systems for dot (signal) counting in fluorescence in situ hybridization (FISH) images have relied on an auto-focusing method for obtaining a clearly defined image. Because signals are distributed in three dimensions within the nucleus and artifacts such as debris and background fluorescence can attract the focusing method, valid signals can be left unfocused or unseen. This leads to dot counting errors, which increase with the number of probes. METHODS: The approach described here dispenses with auto-focusing, and instead relies on a neural network (NN) classifier that discriminates between in and out-of-focus images taken at different focal planes of the same field of view. Discrimination is performed by the NN, which classifies signals of each image as valid data or artifacts (due to out of focusing). The image that contains no artifacts is the in-focus image selected for dot count proportion estimation. RESULTS: Using an NN classifier and a set of features to represent signals improves upon previous discrimination schemes that are based on nonadaptable decision boundaries and single-feature signal representation. Moreover, the classifier is not limited by the number of probes. Three classification strategies, two of them hierarchical, have been examined and found to achieve each between 83% and 87% accuracy on unseen data. Screening, while performing dot counting, of in and out-of-focus images based on signal classification suggests an accurate and efficient alternative to that obtained using an auto-focusing mechanism.

Amniotic Fluid↗
PubMed2001

Hyperspectral imaging: a novel approach for microscopic analysis.

BACKGROUND: The usefulness of the light microscope has been dramatically enhanced by recent developments in hardware and software. However, current technologies lack the ability to capture and analyze a high-resolution image representing a broad diversity of spectral signatures in a single-pass view. We show that hyperspectral imaging offers such a technology. METHODS AND RESULTS We developed a prototype hyperspectral imaging microscope capable of collecting the complete emission spectrum from a microscope slide. A standard epifluorescence microscope was optically coupled to an imaging spectrograph, with output recorded by a CCD camera. Software was developed for image acquisition and computer display of resultant X--Y images with spectral information. Individual images were captured representing Y-wavelength planes, with the stage successively moved in the X direction, allowing an image cube to be constructed from the compilation of generated scan files. This prototype instrument was tested with samples relevant to cytogenetic, histologic, cell fusion, microarray scanning, and materials science applications. CONCLUSIONS: Hyperspectral imaging microscopy permits the capture and identification of different spectral signatures present in an optical field during a single-pass evaluation, including molecules with overlapping but distinct emission spectra. This instrument can reduce dependence on custom optical filters and, in future imaging applications, should facilitate the use of new fluorophores or the simultaneous use of similar fluorophores.

Humans↗
PubMed2001

Velocity estimation of spots in three-dimensional confocal image sequences of living cells.

BACKGROUND: The analysis of three-dimensional (3D) motion is becoming increasingly important in life cell imaging. A simple description of sometimes complex patterns of movement in living cells gives insight in the underlying mechanisms governing these movements. METHODS: We evaluate a velocity estimation method based on intensity derivatives in spatial and temporal domain from 3D confocal images of living cells. Cells of the sample contain intense spots throughout the cell nucleus. In simulations, we model these spots as Gaussian intensity profiles which are constant in intensity and shape. To quantify the quality of the estimated velocity, we introduce a reliability measure. RESULTS: For constant linear velocity, the velocity estimation is unbiased. For accelerated motion paths or when a neighboring spot disturbs the intensity profile, the method results are biased. The influence of the point-spread function on the velocity estimation can be compensated for by introducing anisotropic derivative kernels. The insight gained in the simulations is confirmed by the results of the method applied on an image sequence of a living cell with fluorescently labeled chromatin. CONCLUSIONS: With the velocity estimation method, a tool for estimating 3D velocity fields is described which is successfully applied to a living cell sequence. With the estimated velocity fields, motion patterns can be observed, which are a useful starting point for the analysis of dynamic processes in living cells.

Animals↗
PubMed2001

Automated topographical cell proliferation analysis.

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.

Animals↗
PubMed1993

Variation in DNA content of immature normal bone marrow cells.

A variability in DNA content detected was found with image cytometry, in immature bone marrow cells from 13 healthy donors (median age 31 yr). The mean coefficient of variation (C.V.) of the DNA content was found to be significantly (P = 0.0002) higher in immature blasts and promyelocytes than in mature granulocytes, lymphocytes, and monocytes. The finding was not due to high DNA contents secondary to DNA-synthesis in immature cells, since the percentage of such cells with a reduced DNA content was also significantly (P = 0.0424-0.0002) increased. The error of the method expressed as the variance of multiple measurements has been found to be 0.0002-0.0004. A staining error has been found for some hydrolysis times, but not for times between 60 and 120 minutes. A measuring error was found for an average of 1.38 to 3.38% of the cells. If many normal cells with DNA-aneuploidy are sterile, this would explain the present findings as well as previous ones about intra-marrow cell death, and also the fact that the variability in DNA content could not be detected with cytogenetic or flow cytometric methods.

Adult↗
PubMed1995

3T3 cell motility and morphology before, during, and after exposure to extremely-low-frequency magnetic fields.

Automated image cytometry techniques were used to measure motility and morphology in 3T3 fibroblasts exposed to extremely-low-frequency (ELF) magnetic fields. Cell motility and morphology were measured as a function of time before, during, and after 3-4 hour exposures to vertically oriented, 100 microTRMS sinusoidal magnetic fields at various frequencies in the 10-63 Hz range. Sham exposures were also carried out. No static DC fields were applied, but the geomagnetic field was almost vertical and, therefore, had a large component (28.3 microT) parallel to the applied AC field. The morphology and motile behavior of the cells were characterized by mathematically defined descriptors, which were calculated and averaged for the exposure period as well as for control periods that preceded and followed the exposure period. Each experiment involved the tracking of 100 cells that were subjected to one of the test frequencies (unless a sham exposure was being conducted). Statistical analysis of the results showed that even small changes of 10-20% could be significant at the P < .05 level. Changes on this order were measured in a significant proportion of the experiments. However, because such results were seen for both the sham-exposed and the ELF-exposed cells, and because the range of values that was obtained for the sham exposures was the same as that obtained for the ELF exposures, we concluded that there was no evidence to show that any of the measured changes were attributable to the applied ELF magnetic field.

3T3 Cells↗
PubMed2002

Horizontal cell density and mosaic regularity in pigmented and albino mouse retina.

The present study has examined the density and mosaic regularity of the population of horizontal cells in the pigmented and albino mouse retina. Retinal wholemounts were immunostained for calbindin, and labeled cells within sampled fields were analyzed to determine horizontal cell soma size and density. The X-Y positional coordinates of each cell were determined, from which the geometrical properties of the mosaic were examined using nearest neighbor and Voronoi domain analyses, and regularity indices were derived from those measures. Autocorrelation and density recovery profile analyses were also conducted to identify the presence of exclusion zones within the population of horizontal cells. For each sampled field, random simulations of matched density, constrained by the physical size of the horizontal cells, were generated and analyzed in parallel. Neither retinal area, nor horizontal cell soma size, nor density differed between the pigmented and albino retinas. Mosaic regularity in pigmented and albino retinas did not differ, but each differed significantly from random simulations of identical density. Horizontal cells in the mouse retina exhibit exclusion zones extending beyond the physical size of the soma, but these were identical in size in the pigmented and albino retina. Such exclusion zones are suggested to reflect homotypic interactions between horizontal cells during early development that mediate cellular repulsion and tangential movement. The lack of any discernable effect brought about by the albino mutation, despite numerous developmental abnormalities associated with the retinal neuroepithelium in albino mice, is consistent with other results showing that homotypic interactions are sufficient for the genesis of the global patterning characteristic of mature retinal mosaics.

Albinism↗
PubMed2002

Karyotyping of comparative genomic hybridization human metaphases by using support vector machines.

BACKGROUND: Comparative genomic hybridization (CGH) is a relatively new molecular cytogenetic method for detecting chromosomal imbalance. Karyotyping of human metaphases is an important step to assign each chromosome to one of 23 or 24 classes (22 autosomes and two sex chromosomes). Automatic karyotyping in CGH analysis is needed. However, conventional karyotyping approaches based on DAPI images require complex image enhancement procedures. METHODS: This paper proposes a simple feature extraction method, one that generates density profiles from original true color CGH images and uses normalized profiles as feature vectors without quantization. A classifier is developed by using support vector machine (SVM). It has good generalization ability and needs only limited training samples. RESULTS: Experiment results show that the feature extraction method of using color information in CGH images can improve greatly the classification success rate. The SVM classifier is able to acquire knowledge about human chromosomes from relatively few samples and has good generalization ability. A success rate of moe than 90% has been achieved and the time for training and testing is very short. CONCLUSIONS: The feature extraction method proposed here and the SVM-based classifier offer a promising computerized intelligent system for automatic karyotyping of CGH human chromosomes.

Chromosomes, Human↗
PubMed2002

Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry.

BACKGROUND: Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells. METHODS: The PhiPhiLux-G(1)D(2) caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy. RESULTS: The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation. CONCLUSIONS: Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis.

Animals↗