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PubMed2004

Functional fluo-3/AM assay on P-glycoprotein transport activity in L1210/VCR cells by confocal microscopy.

Multidrug resistance (MDR) phenotype of L1210/VCR cell line, acquired by selection for vincristine (VCR), is predominantly mediated by P-glycoprotein (Pgp). Calcein/AM (Cal) was recently described as a fluorescent substrate for Pgp and may be used for measuring of transport activity of Pgp. Expression of Pgp in the cells prevents them to be loaded with the fluorescent marker. To detect the activity of Pgp, verapamil (Ver) or cyclosporine A (CsA) has to be used as Pgp inhibitors. Multidrug resistance protein (MRP), another drug efflux pump, may be inhibited by probenecid (Pro), i.e, the inhibitor of a wide variety of anion transporters. Ver, but not Pro, is able to induce the loading of L1210/CR cells by Cal that is measurable by fluorescence-activated cell sorter (FACS). Another dye, fluo-3/AM (F-3), has a similar behaviour like Cal. Using confocal microscopy we have proved that L1210/VCR cells, in contrast to parental sensitive cells, are not loaded with F-3. Marking of cells with the dye can be achieved using inhibitors of Pgp like Ver or CsA but not by Pro. These results indicate that F-3 is usable for detection of Pgp function in various MDR tissue cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

[Confocal microscopy study of 2 cases of Thygeson's epithelial keratitis].

Two cases of bilateral Thygeson's keratitis were examined by in vivo confocal microscopy. In the corneas, fine deposits of highly reflected material appeared immediately below the basal lamina. This is the first time that such morphological features are described in patients with Thygeson's keratitis.

Adolescent↗
PubMed1995

Visualization of endothelial clefts and nuclei in living microvessels with combined reflectance and fluorescence confocal microscopy.

OBJECTIVE: The cellular basis for spatial heterogeneity along postcapillary venules in their response to inflammatory mediators is not understood. To study permeability regulatory processes on an individual cell basis in intact microvessels, we developed methods to delineate individual endothelial cells within living, single perfused microvessels. METHODS: Individual postcapillary microvessels in the mesenteries of frogs and hamsters were perfused with chloride-free Ringer's solutions containing AgNO3 (0.1 g per 100 ml) for 5--10 s. Vessels were immediately flushed with Ringer's solution containing serum albumin and some vessels subsequently perfused with the fluorescent nucleic acid stain YO-PRO-1 (1 microM) for 10-15 min. Vessels were imaged in situ using laser scanning confocal microscopy. A reflectance image from silver precipitate in the endothelial clefts and a fluorescence image of the nuclei were simultaneously recorded. Stacks of confocal images were merged and used to reconstruct the three-dimensional orientation of endothelial cells. Hydraulic conductivity of some frog mesenteric venular vessels, measured before and after perfusion with AgNO3, was used to assess the integrity of the vessels. RESULTS: The endothelial cell clefts were delineated with a very fine, readily imaged precipitate of silver. Hydraulic conductivity measured after AgNO3 perfusion in frog mesenteric venular microvessels was not significantly different from control. Vessels showed a slight reduction in reactivity to inflammatory stimuli. The three-dimensional pattern of endothelial cells in the living vessels was imaged. CONCLUSIONS: Combining confocal microscopy with silver staining in single perfused microvessels was shown to be an effective means to delineate the three-dimensional pattern of endothelial cells forming microvessel walls. It enables further study of the vascular structure and function relationships at the individual cell level in intact microvessels.

Animals↗
PubMed2006

Different metabolic responses in alpha-, beta-, and delta-cells of the islet of Langerhans monitored by redox confocal microscopy.

Blood glucose homeostasis is mainly achieved by the coordinated function of pancreatic alpha-, beta-, and delta-cells, which secrete glucagon, insulin, and somatostatin, respectively. Each cell type responds to glucose changes with different secretion patterns. Currently, considerable information can be found about the signal transduction mechanisms that lead to glucose-mediated insulin release in the pancreatic beta-cell, mitochondrial activation being an essential step. Increases in glucose stimulate the mitochondrial metabolism, activating the tricarboxylic acid cycle and raising the source of redox electron carrier molecules needed for respiratory ATP synthesis. However, little is known about the glucose-induced mitochondrial response of non-beta-cells and its role in the stimulus-secretion coupling process. This limited information is probably a result of the scarcity of these cells in the islet, the lack of identification patterns, and the technical limitations of conventional methods. In this study, we used flavin adenine dinucleotide redox confocal microscopy as a noninvasive technique to specifically monitor mitochondrial redox responses in immunoidentified alpha-, beta-, and delta-cells in freshly isolated intact islets and in dispersed cultured cells. We have shown that glucose provokes metabolic changes in beta- and delta-cell populations in a dose-dependent manner. Conversely, no significant responses were observed in alpha-cells, despite the sensitivity of their metabolism to drugs acting on the mitochondrial function, and their intact ability to develop Ca2+ signals. Identical results were obtained in islets and in cultures of dispersed cells. Our findings indicate metabolic differences in glucose utilization among the alpha-, beta-, and delta-cell populations, which might be important in the signal transduction events that lead to hormone release.

Adenosine Triphosphate↗
PubMed1994

Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy.

Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4+ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluorescence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37 degrees C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37 degrees C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1) T = 0, continuous peripheral staining; (2) T = 15 min, patchy peripheral staining; (3) T = 30 min, patches or intracellular vesicular staining; (4) T = 45 min, intracellular vesicular staining; (5) T = 60 min, diffuse intracellular staining; (6) T = 90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed2003

Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry.

PURPOSE: In the epidermis, the highest clonogenicity, a feature of stem cells (SCs), is found in the smallest keratinocyte. In the limbal-corneal (LC) epithelium the SCs are exclusively localized in the basal epithelial layer of the limbal domain. The current study was conducted to determine whether this spatial SC arrangement is reflected in differences in the cell size between limbal and corneal cells. METHODS: In vivo confocal microscopy was used to scan and measure the size of the cells of the central cornea and the superior limbus in five normal subjects, from the superficial to the basal cell layer. Limbal and corneal pure epithelial sheets were isolated by dispase digestion from human tissues and dissociated into single cells by trypsin digestion. The forward (FSC; a relative measure of cell size) and side (SSC; a relative measure of cytoplasmic complexity) light-scattering properties of these cells were determined by flow cytometry. RESULTS: Confocal microscopy showed that diameters of the basal cells of the limbal and corneal zones were 10.1 +/- 0.8 and 17.1 +/- 0.8 micro m, respectively. The corresponding values for the superficial layers were 19.9 +/- 1.6 and 36.6 +/- 1.6 microm, respectively (P < 0.0001). The mean FSC and SSC of the limbal cells amounted to 65.7% +/- 8.7% and of the corneal cells, 74.4% +/- 4.6%. Furthermore, only 1.40% +/- 0.83% and 0.69% +/- 0.37% of the corneal cells had FSC and SSC equal to the lowest 15% of FSC and SCC of the limbal cells, respectively, indicating that the limbus contained a substantial proportion of very low FSC and SSC cells for which there was no corneal counterpart. CONCLUSIONS: The data collectively demonstrate that the smallest cells are located in the limbal basal epithelium. This feature may help isolate corneal SCs located in the limbus.

Adult↗
PubMed1997

[In vivo imaging of corneal innervation in the human using confocal microscopy].

To date, descriptions of the structure of corneal innervation have only been possible on the basis of histological techniques. Confocal microscopy represents a new method for the structural examination of the cornea in vivo. Through our examinations we first defined the control group and then proceeded to record the reinnervation of donor tissue after perforating keratoplasty. We used the confocal slit-scanning video-microscope Microphthal to examine 40 corneas from 20 normal volunteers, 15 donor corneas and 5 eyes after enucleation for ocular tumors. These results were compared to our findings from postoperative checks on 14 patients after perforating keratoplasty (from 1 month to 2 years). With this system we were able to see nerves in the middle and in the superficial stroma. The course of these nerves can be followed, as well as their branching in the subepithelial plexus. Nerve fibers from superficial stromal nerves penetrate Bowman's membrane and create the basal epithelial plexus in the region of the basal epithelium. Seven months after perforating keratoplasty the first stromal nerves could be seen in the central corneal area. The first central reinnervation in the region of Bowman's membrane as well as in the basal epithelium was not detected until 15 months after operation. With confocal microscopy we have the potential to study the morphology of corneal innervation in vivo and in fresh donor tissue. For the first time it is possible to perform non-invasive morphological studies of reinnervation of the human cornea after surgical treatment.

Adolescent↗
PubMed2005

[Confocal microscopy of the cornea in photorefractive surgery].

OBJECTIVE: Corneal refractive surgery involves a range of techniques which have assumed a growing importance during the last few years. The advances of very different surgical methods and the early age of the majority of the patients subjected to surgery, limits our knowledge of the histopathologic changes in the operated corneas. For this reason, the development of any diagnostic tool which can permit us to improve our knowledge of the wound healing process would be of significant value. As we describe in our paper, confocal microscopy enables the assessment of the more important advantages for the study of corneal wound healing after refractive surgery: high magnification, harmless, and "in vivo", providing pathogenic information and lateral and axial definition not achievable with other exploration techniques. CONCLUSION: Confocal microscopy appears to be an essential tool for creating a global concept of quality in corneal refractive surgery.

Cicatrix↗
PubMed2001

Morphological evaluation of Schnyder's central crystalline dystrophy by confocal microscopy before and after phototherapeutic keratectomy.

A 29-year-old man with Schnyder's central crystalline dystrophy was treated with phototherapeutic keratectomy (PTK) in his right eye. Ocular examination revealed abnormal deposits of cholesterol and lipid within the corneal stroma (appearing as crystalline spindle-shaped deposits), high myopia, phakic anterior chamber intraocular lens implantation, and myopic macular degeneration in both eyes. Phototherapeutic keratectomy ablation to a central zone of 7.0 mm and a depth of 96 microm was performed with an Aesculap Meditec MEL-70 excimer laser. Confocal microscopy performed before PTK showed multiple deposits of large, brightly reflective crystalline material extending from the anterior to the mid stroma. The Z-scan curves revealed that the highest density of crystalline deposits was located within the first 140 microm of corneal depth. Six months after PTK, confocal microscopy showed a markedly decreased density of corneal crystalline deposits in the anterior stroma.

Adult↗
PubMed1999

Uptake of reconstituted Na,K-ATPase vesicles by isolated lymphocytes measured by FACS, confocal microscopy and spectrofluorometry.

Na,K-ATPase (EC 3.6.1.37, Na,K-ATPase) is a fundamental vital membrane transport and receptor system which, after biosynthesis, is exported to the plasma membrane in inside-out vesicles. Na,K-ATPase can be extracted form the natural membrane and inserted into artificially formed phosphatidylcholine vesicles (liposomes). The ultrastructure of the reconstituted vesicles has been fully described. In the present work, the Na,K-ATPase-vesicles were labeled with fluorescent tracers either in their water or membrane phase, incubated with freshly isolated human lymphocytes, and the resulting cellular fluorescence measured with fluorescence activated cell sorting (FACS), confocal microscopy and spectrofluorometry. The FACS data show that all lymphocytes take up Na,K-ATPase-vesicles in a dose- and temperature-dependent fashion. Three-dimensional analysis of the fluorescence by confocal microscopy reveals that the fluorescence is contained within the cells. Quantitative determination by spectrofluorometry indicates that depending on the vesicle/cell ratio, a single lymphocyte takes up 650 to 36,500 vesicles within 30 min at 37 degrees C together with up to about 200,000 renal Na,K-ATPase molecules.

Cell Membrane↗
PubMed1995

Detection of primary and mature transcripts of calcitonin-gene-related peptide genes in rat parafollicular cells by light, fluorescence and confocal microscopy.

Alternative splicing of primary transcripts from the calcitonin/alpha calcitonin gene-related peptide (alpha CGRP) gene result in mature mRNAs encoding either calcitonin or alpha CGRP. We have produced sequence-specific, synthetic, biotinylated oligodeoxynucleotide probes that recognize calcitonin (exon 4), and alpha CGRP (exon 6) sequences as well as sequences common to both splice variants (exon 3) of this gene. Probes to exons 4 and 3 revealed strong cytoplasmic signals in rat parafollicular cells. In addition, a punctate nuclear signal was obtained with these probes. The alpha CGRP-specific (exon 6) probe resulted in weak cytoplasmic labelling of parafollicular cells, but produced a punctate nuclear labelling similar to that seen with the exon 4 and 3 probes. RNase digestion removed all the cytoplasmic and nuclear signals obtained with all probes. Hybridization with a thyroglobulin-specific probe failed to label parafollicular cells. A control (human enterovirus) probe yielded negative results, while a probe to rat somatostatin produced cytoplasmic labelling of a small subpopulation of parafollicular cells. Finally, a probe specific for beta CGRP mRNA labelled most, if not all, parafollicular cells. Fluorescent alkaline phosphatase development of in situ hybridizations could be combined with indirect immunofluorescence for CGRP. Analysis by fluorescence and confocal microscopy revealed that CGRP immunoreactive cells contained calcitonin, alpha CGRP and beta CGRP hybridization signals. Our results demonstrate that all three genes may be simultaneously expressed by thyroid parafollicular cells and show that synthetic biotinylated oligonucleotide probes can be used for highly precise localizations of primary transcripts in the nuclei of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Confocal microscopy and stereology: estimating volume, number, surface area and length by virtual test probes applied to three-dimensional images.

The opportunities of confocal microscopy applied to morphometry of microscopical structures are presented and demonstrated on stereological methods based on evaluation of optical sections within a thick slice and using computer-generated virtual test probes. Such methods, allowing arbitrary orientation of the thick slice, can be used for estimating volume, number, surface area, and length. The methods using spatial grid of points, disector, fakir, and slicer probes are described and illustrated by different examples using our freeware 3DTOOLS software and their variance and applicability are discussed. It is shown that shifted triple or quadruple spatial grids of lines are very efficient for the surface area and volume estimation by the fakir method.

Cell Count↗
PubMed1996

Viability analysis of cryopreserved rat pancreatic islets using laser scanning confocal microscopy.

We have developed a digital image analysis technique to assay the viability of frozen-thawed pancreatic islets by using laser scanning confocal microscopy (LSCM) in conjunction with double fluorescent staining [acridine orange/propidium iodide (AO/PI)]. Freshly isolated rat pancreatic islets were cultured for 18-24 h and then brought to a 2 M concentration of dimethyl sulfoxide (Me2SO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at a defined temperature (-10 degrees C), followed by a controlled period for equilibration and then cooling in a programmable bulk freezer at rates of 0.3, 1, 3, 10 and 30 degrees C/min to -70 degrees C. Samples were then stored in liquid nitrogen. Subsequent to rapid thawing and serial dilution with sucrose solution to remove Me2SO, AO/PI-stained individual islets were prepared for imaging on the LSCM. A series of optical sections through individual stained islets were obtained and processed to obtain high-contrast images at two different wavelengths; 488 nm and 514 nm for viable and damaged tissue, respectively. Image analysis algorithms consisted of template masking, generation of histograms of the pixel intensity profile, and gray level thresholding to obtain binary images. The total percentages of both types of tissue in the islet were computed by summing the two populations in each serial section. The spatial distributions of viable and damaged tissue were calculated from the three-dimensional (3-D) data base for both cultured (control) and cryopreserved islets. The 3-D spatial distributions of damaged and viable tissue in the islets were computed by determining the normalized distance of each viable/damaged voxel from the centroid of the islet volume to a mathematically estimated 3-D superquadric surface used to estimate the outer boundary of the islet. Further, each isolated damaged cell was identified and its volume determined. These studies indicate that cryopreserved islets exhibit shape distortion and a decrease in the numerical density of cells in comparison to unfrozen controls. Maximal survival was observed at the slower cooling rates. Accordingly, damage was found to occur throughout the 3-D islet volume in distinct spatial distributions for islets frozen at the slower and the faster cooling rates. Further, it was found that the volume of the majority of damaged cells identified was consistent with that of cells ranging in diameter from 5 to 9 micrometers.

Algorithms↗
PubMedDate not supplied

Application of in vivo confocal microscopy to the understanding of surfactant-induced ocular irritation.

The purpose of this study was to assess the ability of in vivo confocal microscopy (CM) to provide noninvasively derived histopathologic correlates of surfactant-induced eye irritation from which specific pathologic mechanisms can be identified and later evaluated in alternative in vitro models. Rats and rabbits, divided into groups of 5, received 10 microliters of an anionic or cationic surfactant in one eye with the other eye used as a control. At specified times, eyes were examined and scored for ocular irritancy using a penlight and slit-lamp. Subsequently, corneas were evaluated by in vivo CM to evaluate epithelial layer thickness and surface epithelial cell area, corneal thickness, depth of necrosis, inflammation, fibrosis, and endothelial injury. At 3 hr, the anionic surfactant produced slight irritation with peak scores of 12.4 and 8.0 out of a possible 110 in the rats and rabbits, respectively. In vivo CM revealed changes limited to the corneal epithelium that decreased in thickness to 78% in rats and 81% in rabbits at 3 hr. This decrease in the thickness correlated with a significant decrease in surface epithelial cell area from 2,061 +/- 395 microns2 to 567 +/- 330 microns2 in the rats and 1,523 +/- 185 microns2 to 934 +/- 71 microns2 in the rabbits (p < 0.005 and 0.005, respectively). The cationic surfactant produced severe irritation in both the rats and rabbits with peak scores of 85.4 and 80.2 occurring at day 2, respectively. In vivo CM in the rats showed complete loss of corneal epithelium, lysis of keratocytes, and loss of corneal endothelium. In the rabbits, injury appeared limited to the anterior cornea with complete loss of epithelium and loss of keratocytes extending to 52% of the corneal thickness. These findings establish the application of noninvasive, in vivo CM to qualitatively and quantitatively characterize the pathobiology of ocular irritation in situ. This information will be important in the development and evaluation of mechanistically based in vitro alternatives for ocular irritancy testing.

Animals↗
PubMed2006

Noninvasive evaluation of allergic and irritant contact dermatitis by in vivo reflectance confocal microscopy.

BACKGROUND: The clinical differentiation of allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) is often difficult to accomplish. Reflectance-mode confocal microscopy (RCM) is an imaging technique that has previously been used to examine ACD and ICD noninvasively in vivo. OBJECTIVE: To determine characteristic features of ACD and ICD and their kinetic evolution over time. Ethnic susceptibility to contact irritants such as sodium lauryl sulfate and Ivory dishwashing liquid was evaluated noninvasively, and the sensitivity and specificity of RCM parameters were analyzed in a clinical context and in reference to patch testing. METHODS: Subjects were patch-tested with allergens, irritants, and controls. Clinical scoring and RCM evaluation were performed at various time points, assessing stratum corneum (SC) disruption, spongiosis, exocytosis, vesicle formation, and epidermal thickness. RESULTS: RCM features of both ACD and ICD include spongiosis, exocytosis, vesicle formation, and blood vessel dilatation. SC disruption, epidermal necrosis, and hyperproliferation are hallmarks of ICD whereas ACD more typically presents with vesicle formation. Patients with ICD showed a more rapid recovery than those with ACD. When tested with Ivory soap at selected concentrations, Caucasians, when compared to African Americans, showed significantly lower clinical thresholds for ICD and features that were more severe. CONCLUSIONS: RCM may be a promising new technology for longitudinal noninvasive studies of contact dermatitis (CD). Using a diagnostic algorithm and those parameters with high sensitivity for CD, RCM may facilitate the differentiation of acute ACD and ICD. RCM can reliably visualize cutaneous changes at subclinical degrees of CD, which suggests a possible role for RCM as an adjunctive tool in CD diagnosis. The results of this pilot study also indicate ethnic differences in the response to contact irritants. However, further studies are needed to substantiate the relevance and clinical applicability of our findings.

Adult↗
PubMed2003

A practical technique for differentiation of subepidermal bullous diseases: localization of in vivo-bound IgG by laser scanning confocal microscopy.

OBJECTIVE: To develop a practical technique to distinguish autoimmune subepidermal bullous diseases. DESIGN: A prospective study. SETTING: Academic referral center-the Department of Dermatology, Medical University of Warsaw. Patients Forty-two patients fulfilling clinical, immunological, and/or immunoelectron microscopic criteria for bullous pemphigoid (n = 31), mucous membrane pemphigoid (n = 6), or epidermolysis bullosa acquisita (n = 5), diagnosed as having disease and treated from January 1, 1997, to December 31, 2002. MAIN OUTCOME MEASURES: We applied laser scanning confocal microscopy to determine the localization of in vivo-bound IgG at the basement membrane zone in biopsy specimens taken from patients' skin to compare the localization of basement membrane zone markers: antibody against beta4 integrin, antibody against laminin 5, and antibody against type IV collagen. In vivo-bound IgG was visualized by labeling with fluorescein isothiocyanate-conjugated anti-human IgG antibody, whereas basement membrane zone markers were labeled with anti-mouse Cy5-conjugated antibodies. RESULTS: In patients with bullous pemphigoid, in vivo-bound IgG was localized on the epidermal side of laminin 5 and co-localized with beta4 integrin. In patients with mucous membrane pemphigoid, IgG was in vivo bound to the dermal-epidermal junction between localization of laminin 5 and type IV collagen. In patients with epidermolysis bullosa acquisita, in vivo-bound IgG was present on the dermal side of type IV collagen. CONCLUSIONS: Laser scanning confocal microscopy allows precise localization of in vivo-bound IgG in patients' skin and, thus, it is a rapid method for the differentiation of mucous membrane pemphigoid from bullous pemphigoid and epidermolysis bullosa acquisita. This tool is suitable for the routine diagnosis of individual patients and for retrospective studies. This method is of special value in those patients in whom circulating autoantibodies are not detectable.

Adolescent↗
PubMed1996

Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry.

Living yeast cells can be selectively stained with the lipophilic cationic cyanine dye DiOC6(3) in a mitochondrial membrane potential-dependent manner. Our study extends the use of flow cytometric analysis and sorting to DiOC6(3)-stained yeast cells. Experimental conditions were developed that prevented the toxic side effect of the probe and gave a quantitative correlation between fluorescence and mitochondrial membrane potential, without any staining of other membranes. The localization of the fluorochrome was checked by confocal microscopy and image cytometry. The mitochondrial membrane alterations were also tested through cardiolipin staining with nonyl acridine orange. Differences in light scattering and in fluorescence were detected in mutants (rho-, rho degrees, mit-, or pet-) and wild-type (rho+mit+) populations of yeast. The dye uptake of respiratory-deficient yeast strains was significantly reduced as compared to that of the wild-type. Application of an uncoupler (mCICCP), which collapsed the mitochondrial membrane potential (alphapsi(m)), led to a drastic reduction of the dye uptake. It was observed that a decrease in deltapsi(m), was usually correlated with a decrease in cardiolipin stainability by nonyl acridine orange (NAO). Quantitative flow cytometry is a fast and reproducible technique for rapid screening of yeast strains that might be suspected of respiratory dysfunction and/or mitochondrial structural changes. We give evidence that it is an adequate method to characterize and isolate respiratory mutants through sorting procedure, with selective enrichment of the population studied in respiring or non-respiring yeast cells. Confocal microscopy and image cytometry corroborate the flow cytometry results.

Acridine Orange↗
PubMed2005

Non-beating HL-1 cells for confocal microscopy: application to mitochondrial functions during cardiac preconditioning.

HL-1, the first cell line with a cardiac phenotype for biological experiments, displays spontaneous electrophysiological and mechanical regular activity, and cyclic calcium movements. We isolated a derived line, devoid of transient movements, for confocal microscopy experiments. These cells do express cardiac proteins: connexin 43, the cardiac isoform of dihydropyridine receptors, desmin, and developmental myosin but have no sarcomeric arrangement. They still possess the electrophysiological characteristics and ionic currents of cardiac cells, among them the cardiac potassium current IKr. We also found diazoxide and glibenclamide sensitive potassium channels with properties similar to IK(ATP) in adult cardiac myocytes. The pacemaker current I(f) was not observed, in agreement with the cells showing excitability but lacking in pacemaker activity. The absence of movement is an advantage for studies which include changes of media in order to follow morphological changes under continuous perfusion. We observed however a basal spontaneous movement of mitochondria and we developed a method to quantify its amplitude using confocal microscopy. No mitochondrial depolarization could be detected when the membrane potential was measured by using very low light photomultiplier and confocal fluorescence imaging under the K(ATP) channel opener diazoxide. Thus cardiac pharmacological preconditioning by K(ATP) channel openers might involve other routes than mitochondrial K channels targeting.

Adenosine Triphosphate↗