Wavelet transform analysis of chromatin texture changes during heat shock.

Texture analysis can be a useful tool to investigate the organization of chromatin. Approaches based on multiscale analysis and in particular the 'à trou' wavelet analysis has already been used for microscopy (Olivo Marin). In order to analyse texture changes, the statistical properties of the wavelet coefficient images were summarized by the first four statistical orders: mean, standard deviation, skewness and kurtosis of the coefficient image histogram. The 'à trou' transform provided a representation of the wavelet coefficients and texture parameters with the same statistical robustness throughout the scale spaces. It was applied for quantifying chromatin texture and heat-induced chromatin changes in living cells. We investigated the changes by both laser scanning and spinning disk confocal microscopies and compared the texture parameters before and after increasing duration of heat shock exposure (15 min, 30 min and 1 h). Furthermore, as activation of the heat shock response also correlates with a rapid localization of HSF1 within a few nuclear structures termed nuclear stress bodies (nSBs), we compared the dynamics of nSBs formation with that of textural changes during 1 h of continuous heat shock. Next, we studied the recovery phase following a 1-h heat shock. Significant differences were observed, particularly affecting the perinucleolar region, even for the shortest heat shock time affecting mostly the skewness and standard deviation. Furthermore, progressive changes could be observed according to the duration of heat shock, mostly affecting fine details (pixel-wise changes) as revealed by the parameters, obtained from the first- and second-order wavelet coefficients. 'A trou' wavelet texture analysis provided a sensitive and efficient tool to investigate minute changes of chromatin.

Continuous photobleaching in vesicles and living cells: a measure of diffusion and compartmentation.

We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime (t<0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime (t>0.1-5 s), for which the diffusion and the photobleaching balance each other. In this long time regime, the diffusion is either fast (i.e., the photobleaching probability of a molecule diffusing through the laser beam is low) so that the photobleaching rate is independent of the diffusion constant and dependent only of the laser power, or the diffusion is slow (i.e., the photobleaching probability is high) and the photobleaching rate is mainly dependent on the diffusion constant. We illustrate our theory by using giant unilamellar vesicles ranging from approximately 10 to 100 microm in diameter, loaded with molecules of various diffusion constants (from 20 to 300 microm2/s) and various photobleaching cross sections, illuminated under laser powers between 3 and 100 microW. We also demonstrated that information about compartmentation can be obtained by this method in living cells expressing enhanced green fluorescent proteins or that were loaded with small FITC-dextrans. Our quantitative approach shows that molecules freely diffusing in a cellular compartment do experience a continuous photobleaching. We provide a generic theoretical framework that should be taken into account when studying, under confocal microscopy, molecular interactions, permeability, etc.

Photobleaching, mobility, and compartmentalisation: inferences in fluorescence correlation spectroscopy.

In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter P(B) = sigma(B)P/D (where sigma(B) is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when P(B) << 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like sigma(B)P/R2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.

Confocal image characterization of human papillomavirus DNA sequences revealed with Eu in HeLa cell nuclei stained with Hoechst 33342.

OBJECTIVE: To visualize and localize specific viral DNA sequences revealed with Eu by fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM) and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Human papillomavirus DNA (HPV-DNA) was identified in HeLa cells with biotinylated DNA probes recognizing HPV-DNA types 16/18. DNA-DNA hybrids were revealed by a three-step immunohistochemical amplification procedure involving an antibiotin mouse monoclonal antibody, a biotinylated goat antimouse polyclonal antibody and streptavidin-Eu. Cell nuclei were counterstained with Hoechst 33342. Image sequences were obtained using a CLSM that made possible ultraviolet excitation. The location of fluorescent signals inside cellular preparations was determined by FAMIS and selection of filters at emission. Image sequences were summarized into a reduced number of images, or factor images, and curves, or factors. Factors estimate spectral or temporal patterns and depth emission profiles. Factor images correspond to spatial distributions of the different factors. RESULTS: We distinguished between Eu corresponding to HPV-DNA hybridization signals and nuclear staining by taking into account differences in their spectral and temporal patterns and (using their decay rates). CONCLUSION: FAMIS, together with CLSM and Eu, made possible the detection and characterization of viral papillomavirus DNA sequences in HeLa cells.

Confocal analysis of phosphatidylserine externalization with the use of biotinylated annexin V revealed with streptavidin-FITC, -europium, -phycoerythrin or -Texas Red in oxysterol-treated apoptotic cells.

OBJECTIVE: To analyze externalization of phosphatidylserine via annexin V on apoptotic cells by laser scanning confocal microscopy and factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Streptavidin-fluorescein isothiocyanate (FITC), -europium (Eu), -phycoerythrin (PE) and -Texas Red (TR) were chosen to reveal the binding of biotinylated annexin V on apoptotic U937 human leukemic cells and ECV-304 human endothelial cells induced under treatment with 7-ketocholesterol or 7 beta-hydroxycholesterol. Excitation of each fluorochrome was obtained by selection of specific lines (351 + 364 nm, 488 nm) of the argon laser of a confocal microscope. Temporal and spectral series were performed to characterize each fluorochrome. FAMIS was applied to these series to estimate images corresponding to stains. RESULTS: Each fluorochrome was clearly distinguished, and images showed localization of phosphatidylserine, which was improved by image analysis. CONCLUSION: On apoptotic cells it is possible to analyze differences in the improved visualization of phosphatidylserine in series processed by FAMIS with the use of biotinylated annexin V revealed with streptavidin-FITC, -Eu, -PE or -TR.

Higher concentrations of histone macroH2A in the Barr body are correlated with higher nucleosome density.

Histone macroH2A, which is a subtype of histone H2A, possesses a histone H2A-like portion fused to a relatively long non-histone portion. MacroH2A has been shown to associate preferentially with the inactive X chromosome [1]. To investigate the specificity of this association, the nuclear distribution of macroH2A was compared with that of regular core histones. In normal human female fibroblasts, all anti-histone antibodies that were tested (including anti-macroH2A antibody) preferentially labeled the inactive X chromosome. Moreover, when expressed as green fluorescent protein (GFP) fusions, both histone H2A and macroH2A were concentrated in the Barr body. These data clearly show the presence of a higher density of nucleosomes in the inactive X chromosome. Accordingly, the specificity of the macroH2A association with the inactive X chromosome should be reconsidered. While investigating the role of macroH2A, we found that the proximity of the non-histone region of macroH2A to a promoter could lead to a specific repression of transcription, suggesting that the incorporation of macroH2A into chromatin might help to establish the stable pattern of gene expression in differentiated cells.

The first case of new variant Creutzfeldt-Jakob disease in France: clinical data and neuropathological findings.

Clinical data and autopsy findings in a case of new variant Creutzfeldt-Jakob disease (vCJD) are reported. This case, the first histologically confirmed case described outside the United Kingdom, very much resembles the cases described by Will et al. [(1996) Lancet 347:921-925] and Zeidler et al. [(1997) Lancet 350:903-908, 908-910]. Neuropathological studies failed to reveal any conspicuous clues that could be relevant for understanding the pathophysiology of the disease. For epidemiological surveillance, neuropathologists should scrutinize suspected cases keeping in mind the possibility of vCJD.

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations.

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.

Automatic enumeration of adherent streptococci or actinomyces on dental alloy by fluorescence image analysis.

The aim of the present study was to develop an automated image analysis method to quantify adherence of Streptococcus sanguinis or Actinomyces viscosus on surfaces of a currently used dental alloy. Counting such bacterial strains was difficult because of their arrangement, thus S. sanguinis being a coccus arranged in chains or pairs, and A. viscosus a long complexly arranged polymorph rod. Direct counting of fluorescently stained adherent bacteria was done visually and with image analysis methods. To differentiate these two morphotypes, two programs were developed: (i) for streptococci, thresholding and selection of the object maxima, and (ii) for actinomyces, two step thresholding and processing of the characteristic points of the object skeletons. The triplicate enumerations for each bacterial strain were not significantly different (p > 0.005) and correlations between visual counting and automated counting were significant (r = 0.91 for S. sanguinis and r = 0.99 for A. viscosus, p <00.0001). These rapid and reproducible methods, allowed us to count either cocci or rods, adherent on an inert substratum, in high density conditions.

An image analysis method for the study of cell adhesion to biomaterials.

This fluorescence image analysis method for the quantitative determination of cell adhesion on biomaterials allows bone cells labelled with propidium iodide to be counted automatically, directly on their support. The reliability of the estimation by fluorescence image analysis was validated by comparison with visual counting and with results obtained by an electronic particle counter. In this way it was possible to demonstrate that the adhesive properties of bone cells are dependent on the type of substrate--enstatite (MgO, SiO2, CaO-P2O5-Al2O3), Thermanox (modified polyethyleneterephthalate), or glass. In contrast, the spread of the cell cytoplasm, labelled with fluorescein isothiocyanate and measured by image analysis, does not vary significantly according to the substrate. The characterisation by SKIZ tessellation of the spatial cell arrangement shows that the bone cells have a random organisation on Thermanox and glass, whereas they form aggregates on enstatite.

Measurement accuracy in confocal microscopy.

Confocal laser scanning microscopy provides optical serial sections through thick biological samples, making it possible to perform both three-dimensional visualization and three-dimensional quantitative analysis. On human lymphocytes, we measured geometrical features, cell contents in DNA and in cyclin A and CDK1 proteins, localization and colocalization of these two proteins. Cells were acquired at a vertical sampling step of 0.5 micron, which gives sufficient information about cell labelling. For the purpose of obtaining fast and reliable data at a reduced time cost, we examined various possibilities to simplify acquisition. For example, it might be possible to increase the vertical sampling step to 2.0 microns while preserving an acceptable accuracy of measurements. Further limiting the acquisition to the central sections appeared to give only rough estimations about the whole cells. Finally, we compared confocal microscopy to conventional two-dimensional epifluorescence microscopy. Confocal microscopy appeared slightly less accurate as regards content estimation, but was an invaluable tool when investigating three-dimensional structures and, more especially, localization of proteins.

Cell migration and MMP-9 secretion are increased by epidermal growth factor in HaCaT-ras transfected cells.

Mutated RAS oncoproteins and epidermal growth factor (EGF) are thought to contribute to the proliferative, invasive and metastatic properties of transformed cells. In the present study, we investigated the role of EGF in two H-ras transfected clones and compared it to that in the parental cell line, HaCaT and primary cultured keratinocytes. Our findings show that the motility on type I collagen, measured by the migration index, was similar for both the HaCaT cell line and normal human keratinocytes, whereas it was higher for the HaCaT-ras clones. These results suggest an involvement of the ras oncogene in the stimulation of cell migration. EGF in cell pretreatment or during the migration assay also caused an increase in migration of all the cells, but preserved the difference between HaCaT and HaCaT-ras. However, no significant difference in EGF-R expression was detected between normal cultured keratinocytes, HaCaT and HaCaT-ras cell lines with or without EGF pretreatment. Moreover, when the cells were stimulated with EGF, the MMP-9 activity was greatly increased in a dose-dependent manner in all the cells, and EGF stimulation particularly highlights the increased amount of MMP-9 in HaCaT-ras cells compared to HaCaT cells. In conclusion, EGF is able to enhance motility and to up-regulate MMP-9 activity in all cells, but with a higher impact in HaCaT-ras cells without an overexpression of EGF-R. As EGF acts in synergy with the H-ras mutation, they could be implicated in the local invasion by the HaCaT-ras clones.

Laser scanning confocal imaging of abnormal or arrested human preimplantation embryos.

PURPOSE: The improved resolution and optical sectioning of a confocal microscope make it an ideal instrument for extracting three-dimensional information, especially from extended biological specimens such as human embryos. The staining of actin together with chromatin allowed us to specify the architecture of the embryo and the appearance of the nucleus. METHODS: F-Actin and chromatin distributions were visualized using laser scanning confocal microscopy in "fresh" and "cryopreserved" human preimplantation embryos obtained by in vitro fertilization. RESULTS: The current study revealed a high rate of multinucleation in arrested or poor-quality embryos (89%, in grade IV embryos). CONCLUSIONS: Confocal microscopy revealed high levels of multinucleated blastomeres, suggesting that the probable cause of arrested development in these embryos was due to multinucleation of blastomeres.

Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos.

Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilization. Observation of the cellular actin cortex and chromatin showed a high frequency of embryos with blastomeres exhibiting two or more nuclei, while others had nuclei displaying chromatin condensation and fragmentation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigated using two detection methods for cells undergoing apoptosis. Detection of phosphatidylserine exposure was performed using annexin V; the chromosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which continued to develop normally after thawing. The TUNEL assay was positive in 30% (15/50) of arrested embryos, all of which had cytoplasmic fragments. In contrast, embryos showing regular size blastomeres without fragments were TUNEL negative.

Detection of low copy numbers of HPV DNA by fluorescent in situ hybridization combined with confocal microscopy as an alternative to in situ polymerase chain reaction.

In genital lesions infected by human papillomavirus (HPV), histological criteria and HPV DNA typing are of prognostic value. Therefore, non-radioactive methods such as in situ hybridization are used extensively since they preserve the histological organization of the tissue, and allow the detection and characterization of HPV DNA. However, the sensitivity of these methods is often limited to detection of low copy numbers of HPV DNA in isolated cells or in tissue sections, and therefore alternative techniques have been explored. In the present study, 1-2 copies of HPV DNA were visualized in SiHa cells either by in situ amplification of nucleic acid sequences with the polymerase chain reaction (PCR) or by fluorescent in situ hybridization (FISH) associated with observation by laser scanning confocal microscopy (LSCM). The latter procedure was evaluated for use on histological tissue sections to identify low copy numbers of HPV DNA. Genital lesions which were negative by enzymatic in situ hybridization and FISH but histologically suspected of HPV infection were investigated, and intense signals were obtained both with in situ PCR and with the combined use of FISH and LSCM. Therefore, the combination of FISH with LSCM examination may be as valuable as in situ PCR to detect viral genes present in small amounts in isolated cells and in tissue sections.

Detection of human papillomavirus DNA in genital lesions by enzymatic in situ hybridization with Fast Red and laser scanning confocal microscopy.

Human papillomavirus (HPV) infection with potentially oncogenic types 16 or 18 is common in genital lesions especially in uterine carcinomas. In such lesions, in situ hybridization with non-radioactive probes is a powerful tool for the histopathologist to detect and type HPV DNA either on cell deposits or on tissue sections. The use of an immunohistochemical method involving alkaline phosphatase and Fast Red TR salt/naphthol AS-MX phosphate is proposed for use with conventional bright-field or fluorescence microscopy as well as by laser scanning confocal microscopy. The alkaline phosphatase-Fast Red reaction has the advantage of producing a red precipitate that permits the detection of in situ hybridization signals by bright-field microscopy, and of obtaining a strong red fluorescence characterized by a lack of bleaching when excited by a green light. Therefore, the alkaline phosphatase-Fast Red reaction is well adapted for observations by fluorescence and confocal microscopy, the latter method allowing the detection, in tissue sections of cervical intraepithelial lesions, of small punctate and large diffuse hybridization signals, considered as integrated and episomal states of HPV DNA respectively. The combination of in situ hybridization with the alkaline phosphatase-Fast Red reaction and confocal microscopy is particularly convincing when hybridization signals are of small size and/or of low fluorescence intensity, especially if they are present in various focal planes; in such conditions, infected cells are easily detected by three-dimensional reconstruction. Therefore, this combination is a suitable method for identifying and characterizing HPV DNA in cells and tissue sections.

Preferential expression of kin, a nuclear protein binding to curved DNA, in the neurons of the adult rat.

The KIN17 gene product has been identified by cross immunoreactivity with anti-RecA antibodies and by DNA recombination techniques, and is probably part of the DNA recombination-repair machinery. Following Western blotting and immunocytochemistry using anti-RecA antibodies, and in situ hybridization with specific KIN17 cDNA probes, we here report the detection of high levels of KIN protein and KIN17 mRNA in the CNS of adult rats. The RecA cross-reacting protein has an apparent molecular weight of 41 kDa and is located in the nucleus of brain cells. Both the KIN17 transcript and the protein were found to be widespread, but they were present in different proportions, depending on the type of brain cells. High levels of KIN protein were seen in neurons of the motor nuclei of the brainstem, the locus coeruleus, hippocampal formation, entorhinal cortex, Purkinje cells, pyramidal cells of the cortex and mitral cells. In contrast, using a combination of KIN17 mRNA in situ hybridization and GFAP immunocytochemistry (a marker of glial cells) showed that the KIN17 messenger is preferentially transcribed in neurons, the post-mitotic and long lived brain cells. We postulate that KIN17 play a role in the illegitimate recombination of DNA sequences and/or the repair of alterations of the genome in neurons.

Applications of laser scanning confocal microscopy to the observation of human oocytes and embryos.

Laser scanning confocal microscopy (LSCM) is a powerful and useful tool in developmental biology. The diverse applications of LSCM in biomedical field has led to advances in the microscopes themselves and the synthesis of novel specific probes for the observation of biological structures and the hypothesis of physiological process. LSCM was used to visualize the cellular actin cortex together with the chromatin in human arrested preimplantation embryos and in unfertilized oocytes obtained by in vitro fertilization. In embryos, we observed a majority of multinucleated blastomeres and some with fragmented nuclei or anucleated. LSCM also showed nuclei displaying the chromatin condensation and fragmentation patterns suggesting the phenomenon of apoptosis. Moreover, in arrested fragmented embryos of poor morphology, unequal sized blastomeres often showed cellular fragments without nuclei, compatible with aspects of apoptotic bodies.

Presence of somatostatin in normal human epidermis.

Somatostatin (SOM) is a ubiquitous peptide which is responsible for the inhibition of numerous biological functions. SOM is described as an antiproliferative molecule and an inhibitor of exocrine or endocrine secretion from a variety of tissues, including pancreas, gastrointestinal tract, central and peripheral nervous system. Mediation of SOM effects can be indirect or direct, respectively, through other molecules or receptors on target cells. We have searched for the presence of SOM in the epidermis using immunofluorescence, confocal laser scanning microscopy, radioimmunoassay, and chromatography. Immunofluorescence and confocal laser scanning microscopy studies were performed using rabbit antiserum anti-SOM and mouse monoclonal antibody directed to CD1a Langerhans cell (LC) marker disclosed with fluorescein or tetramethylrhodamine isothiocyanate conjugates. SOM was extracted from whole skin or epidermal cell suspension or LC-enriched suspensions and analysed by radioimmunoassay. We used an antiserum which was reactive for the 6-11 portion of native SOM. Chromatographic columns were performed on extracts from whole skin. The epidermis was SOM immunoreactive. LC were immunoreactive for SOM and the staining was membranous. SOM was extracted from the whole skin at about 0.13 +/- 0.02 fmol/mg of tissue (mean +/- SEM). The SOM concentration in epidermal cell suspensions was 1.5 +/- 0.9 fmol/10(6) cells. Data obtained with LC-enriched suspensions showed large variations between donors. Extracts from skin showed one peak with an elution profile like that of 14 amino acid SOM. This study demonstrates that 14 amino acid SOM is expressed in normal human epidermis.

Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy.

Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by the biotin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was added. Cell fluorescence emission intensity and bleaching characteristics were measured. Rhodol Green exhibited the highest fluorescence intensity and the best photobleaching resistance. Although BODIPY also resisted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence intensity and a fast decay of fluorescence. Among the green emitting fluorochromes tested, Rhodol Green appeared to be the best.