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International audience; BACKGROUND: Environmental surveillance is important in high-risk areas of hospitals to prevent fungal infections in immunosuppressed patients. Conventional culture methods for enumerating environmental fungi are time-consuming. AIM: In this field study, a solid-phase cytometry technique (SPC) and a more conventional culture-based method to quantify fungal contamination of hospital air and surface samples were compared. METHODS: For the air sampling, a liquid cyclone air sampler was used with a flow rate of 300 L/min for 10 min in each of four hospital locations. Surface swabbing was done in two locations, with two different swab types. Samples from all areas were processed by SPC and by culture on malt extract agar. FINDINGS: The mean airborne concentrations of viable fungi determined by SPC were about 1.5-fold higher than the mean concentrations obtained with the culture-based method. These differences for air samples were significant in three hospital environments. No significant difference was observed for surface samples between the two swab types and between the two analytical methods. One of the prominent advantages of SPC was its rapidity in comparison with the culture-based method (5 h versus 5 days). CONCLUSION: In this study, we showed that SPC allows for rapid monitoring of viable fungi in hospital environments. SPC can thus be used to provide an early warning and a rapid implementation of corrective measures. Viable fungi detection may be an important tool to assess infectious risk in wards with immunosuppressed patients.

A task force of experts in the field of diagnostic DNA image cytometry, invited by the ESACP, and further scientists or physicians revealing experience in that diagnostic procedure (names are given in Addendum A), agreed upon the following 4th updated Consensus Report on Standardised Diagnostic DNA Image Cytometry during the 7th International Congress of that society in Caen, 2001. This report is based on the three preceding ones [6,14,17]. It deals with the following items: – Critical review and update of the definitions given in the 1997 Consensus Update; – Review and detailed description of basic terms, principles and algorithms for diagnostic interpretation; – Recommendations concerning diagnostic or prognostic applications in specific fields of tumour pathology. This update is not aimed to substitute the 1997 consensus, but to make necessary addenda and give more detailed descriptions of those items not unequivocally to interpret by potential users of the methodology.

International audience; Quantification of cardiomyocyte contraction is usually obtained by measuring globally cell shortening from the displacement of cell extremities. We developed a correlation-based optical flow method, which correlates the whole-cell temporal pattern with a precise quantification of the intracellular strain wave at the sarcomeres level. A two-dimensional image correlation analysis of cardiomyocytes phase-contrast images was developed to extract local cell deformations from videomicroscopy time-lapse sequences. Test images, synthesized from known intensity displacement fields, were first used to validate the method. Intracellular strain fields were then computed from videomicroscopy time-lapse sequences of single adult and neonatal cardiomyocytes. The propagation of the sarcomeres contraction-relaxation wave during cell contraction has been successfully quantified. The time-varying patterns of intracellular displacement were obtained accurately, even when cardiomyocyte bending occurred in pace with contraction. Interestingly, the characterization of the successive 2D displacement fields show a direct quantification of the variation with time of intracellular strains anywhere in the cell. The proposed method enables a quantitative analysis of cardiomyocyte contraction without requiring wave tracking with the use of fluorescent calcium probes. Thus, our algorithmic approach provides a fast and efficient tool for analyzing the correlation between global cell dynamical behavior and mechanosensitive intracellular processes.

Estrogens influence CNS development and a broad spectrum of neural functions. Several lines of evidence also suggest a neuroprotective role for estrogen. Different modes of estrogen action have been described at the cellular level involving classical nuclear estrogen receptor (ER)-dependent and nonclassical membrane ER-mediated rapid signaling. We have previously shown that nonclassical estrogen signaling is implicated in the control of dopamine cell function and protection. Since nonclassical interactions between estrogens and glia may contribute to these effects, our aim was to demonstrate the presence of membrane-associated ERs and their putative coupling to intracellular signaling pathways in astrocytes. Confocal image analysis and fluorescence-activated cell sorting (FACS) studies indicated the attachment of ER-alpha but not ER-beta to the plasma membrane of astrocytes. ERs were located in the cell soma region and glial processes. FACS analysis revealed that only a subpopulation of midbrain astrocytes possesses membrane ER-alpha. In FACS studies on ER-alpha knockout astrocytes, only a few membrane ER-positive cells were detected. The activation of membrane ERs appears to be coupled to the MAP-kinase/Src signaling pathway as shown by Western blotting. In conclusion, our data provide good evidence that nonclassical estrogen action in astrocytes is mediated by membrane ER-alpha. The physiological consequence of this phenomenon is not yet understood, but it might have a pivotal role in estrogen-mediated protective effects on midbrain dopamine neurons.

To characterize the behavior of magnetofluorescent products injected in mice intravenously.The magnetic resonance imaging (MRI) products were labelled with fluorescent molecules to examine the biodistribution process in vivo and observe them at the cellular level by means of confocal microscopy. Three-dimensional (3D) sequences of images were obtained by spectral analysis of sample preparations in a multiphoton confocal microscope and analyzed by the factor analysis of medical image sequence algorithm, which provides factor curves. Factor images are the result of image-processing methods that utilize information from emission spectra. Preparations are also screened in the counting mode to provide fluorescent lifetime imaging microscopy (FLIM) characterizations.Factor images and FLIM images can help to analyze MRI targeting inside the liver and thoracic aorta of mice. They show positive detection of Fe-Texas red and BOPTA-Eu in the liver and positive detection of Fe-Texas red and negative detection of BOPTA-Eu inside the thoracic aorta.This investigation established the utility of fluorescent MRI contrast agents as in vivo staining tools for cellular sites.

BACKGROUND: Time-lapse video microscopy was used to determine whether mitochondrial and nuclear changes (decrease in mitochondrial transmembrane potential, condensation, and/or fragmentation of the nuclei, morphologic features typical of apoptosis) occurring during 7-ketocholesterol-induced cell death on A7R5 rat smooth muscle cells took place before or after the loss of cell adhesion. In addition, changes in actin organization were followed by conventional fluorescence microscopy. METHODS: Morphologic, functional, and spatial changes at the mitochondrial level were investigated with 3,3'-dihexyloxacarbocyanine iodide and/or MitoTracker Red, and nuclear morphology was characterized by staining with Hoechst 33342. Actin fibers, which are major components of the filament network of the cytoskeleton, were visualized with phalloidin linked to fluorescein. The numbers of adherent and nonadherent cells were determined by cell counting. RESULTS: 7-Ketocholesterol-induced cell death was associated with a rapid alteration of actin fibers, a loss of intercellular junctions, and cell shape modifications. Analysis of mitochondrial transmembrane potential showed successively a hyperpolarization and a more or less pronounced progressive decrease followed by a dramatic drop associated with an increase in Hoechst 33342 staining, reflecting chromatin condensation and morphologic changes in the nuclei. CONCLUSIONS: During cell death induced by 7-ketocholesterol in A7R5 rat smooth muscle cells, the different methods of microscopy allowed us to establish that alterations of actin fibers and mitochondrial dysfunctions occurred before condensation and/or fragmentation of the nuclei, which preceded the loss of cell adhesion.

Background The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. Methods After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. Results Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. Conclusions The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units. Cytometry 43:110–116, 2001. © 2001 Wiley-Liss, Inc.

An increasing need for flexible consultation between pathologists, including the application of fast evolving supplementary technologies, has been identified during the last years. Although pathology is already one of the most advanced application of telemedicine there is more to come from the fast evolution towards computerized microscope image analysis: A reproducible quantification of measurable descriptors of the lesions in cells and tissues (so‐called biological markers) is an indispensable adjunct to routine diagnostic application. Among such quantitative methods DNA image cytometry is increasingly applied by pathologists for assistance in diagnostics.As for other pathological issues, too, a reference center for the clinical application of DNA image cytometry might be therefore of utmost value for pathologists using that method.Based on advanced telematic technologies, a Virtual Reference and Certification Center (VRCC) could be installed for certifying the cytometry hardware and software, the analytical procedures, and the basic interpretation of the results. It will be designed to be operated as a non‐attended service, based on quantification servers accessible via Internet round the clock. The VRCC will supply appropriate standardization and normalization materials and run a GroupWare platform for consensus making by experts.

We report here the study of the biocompatibility of a bone graft material, the Pyrost ® , using a previously established in vitro model of human osteoblasts. The effect of this material on cell proliferation was evaluated by the MTS assay. Results indicated the absolute absence of cytotoxic or cytostatic effect of Pyrost ® on cultured osteoblasts. Viability rate was more than 90% in cells cultured with the material compared to the control. Morphological analysis, undertaken by scanning electron microscopy showed a good adhesion and a spreading of osteoblasts in contact with the material that was colonized by cultured cells. In the second part of this work, we have introduced two methods as complementary biocompatibility tests: DNA image cytometry and interphase Ag-NORs quantification. DNA content was measured in cells cultured with or without Pyrost ® for 3, 9, 15 and 30 days. The determination of DNA indicated that the majority of osteoblasts population was diploid without aneuploidy. The DNA index and cell distribution profile in DNA histograms were similar in all cell populations. The Ag-NORs amount was used as a parameter for cell kinetic evaluation. We have measured the Ag-NORs index like DNA quantification. The proliferation rate, evaluated by Ag-NORs counts in osteoblasts cultured with or without the material, was identical. However, a decrease in Ag-NORs index was observed from day 3 to day 15 of incubation. These results showed a satisfactory biocompatibility of the Pyrost ® in human osteoblasts culture. The material did not alter cell viability and had no inducing effect either on proliferation rate or on cell ploidy as demonstrated by DNA image cytometry and Ag-NORs proteins staining.