Viability Probe Research Articles

Structural and functional cellular alterations underlying the toxicity of methamphetamine in rat retina and prefrontal cortex.

The consumption of illicit drugs is an increasing problem in contemporary societies, and is one of the major causes of death and illness all over the world. Methamphetamine is among the drugs more widely used. Although evidence for a role of reactive species--especially reactive oxygen species (ROS) and apoptotic events--has been shown, the mechanism(s) underlying the cellular toxicity induced by this drug is not yet fully identified. In this context the elucidation of the cytotoxic effects induced by methamphetamine in rat frontal cortex and retina, which compromise cell viability and ultimately result in cell death, can further contribute to the understanding of its mechanism of action. This knowledge may provide new insights into the development of new therapeutic approaches to prevent or ameliorate deleterious alterations of the nervous system. The use of epifluorescence microscopy associated with different fluorescent probes, markers of structural and/or functional cell parameters, can be used as a powerful tool to carry out those studies, in particular, the viability probes propidium iodide (PI) to assess plasma membrane integrity and fluorescein diacetate (FDA), which can monitor intracellular esterase activity and/or pH. In a preliminary study, the kinetic assessment of cellular changes induced by different drug concentrations (0, 1.2, 3, and 6 mM) allowed detection of dose-dependent alterations that are observed earlier in the retina. In fact, in the retina it was possible to monitor alterations (at 4 h of incubation) both in plasma membrane integrity and in esterase activity and/or pH for the lowest drug concentration (1.2 mM). In the prefrontal cortex these changes were only visible for drug concentrations > or = 3 mM. This work is a novel approach to the mechanisms of action of illicit drugs in the central nervous system and will provide the foundations and guidelines for further investigations in the context of tolerance, dependence, and addiction.

Efficacy Enhancement of Trisodium Phosphate against Spoilage and Pathogenic Bacteria in Model Biofilms and on Adipose Tissue

A two-step approach for enhancing the efficacy of trisodium phosphate (TSP) was evaluated using meat spoilage and pathogenic bacteria in flow cell biofilms and adipose tissue model systems. The process was based on the plasmolysis of attached bacteria (biofilms) with a hyperosmotic solution (1.5M NaCl) and the subsequent deplasmolysis of cells with a low-osmotic-strength solution containing different concentrations of TSP (0.1, 0.25, 0.5, 0.625, and 1.0% [wt/vol]). Escherichia coli, Salmonella Enteritidis, Pseudomonas sp., Listeria monocytogenes, and Brochothrix thermosphacta strains were cultivated for 24 h as pure culture biofilms in glass flow cells with complex media and were then treated with either 0.1, 0.25, 0.5, 0.625, and 1.0% TSP, or the same TSP concentrations delivered in conjunction with plasmolysis-deplasmolysis (PDP). Confocal scanning laser microscopy, a commercial fluorescent viability probe, and image analysis were then used to quantify the relative abundances of living and dead cells remaining after the different treatment regimes. With the exception of L. monocytogenes (which was resistant to TSP concentrations of up to 5%), the PDP process increased the sensitivity of the test strains to TSP. However, when similar experiments were conducted with pork adipose tissue, it became evident that higher TSP concentrations were necessary to produce significant decreases in the number of viable cells and that the PDP process generally failed to enhance TSP efficacy. An exception was L. monocytogenes, which exhibited an increase in sensitivity to TSP when inoculated tissue was pretreated with 1.5M NaCl. It is thought that factors contributing to the failure of the PDP process to enhance the activity of TSP in meat systems involves the mode of TSP antimicrobial activity, alkaline pH stress, and the chemically complex, buffered nature of meats. It remains to be determined whether the PDP process is suitable for use with other food grade antimicrobial agents or can be used in nonfood biofilm control applications.

Effect of Bacteriocins Pediocin PD-1, Plantaricin 423, and Nisin on Biofilms ofOenococcus oenion a Stainless Steel Surface

The effect of pediocin PD-1, plantaricin 423, and nisin was tested against established biofilms formed by a commercial starter culture of <i>Oenococcus oeni</i> on stainless steel slides and planktonic (free-living) cells. The percentage viable and nonviable cells were determined by using a viability probe and an epifluorescence microscope with image analysis. Pediocin PD-1 (3000 AU/mL) removed all cells in a biofilm of <i>O. oeni</i> that formed in acidic grape medium after five hours. Plantaricin 423 (3000 AU/mL) and nisin (3000 AU/mL) killed all viable cells in the biofilm. However, approximately 42 and 49% of the original cells on the stainless steel surface remained attached, with the majority staining nonviable when treated with the latter two peptides, respectively. In a modified Chardonnay must, all cells (viable and nonviable) of <i>O. oeni</i> in the biofilm were removed after one hour when treated with pediocin PD-1 (3000 AU/mL). In similar experiments, treatment with plantaricin 423 and nisin resulted in undetectable numbers of viable cells in the biofilm. Approximately 36 and 43% of the original cells on the stainless steel surface remained attached, with the majority staining nonviable after five hours of treatment with the latter two peptides, respectively. After five hours of treatment with the respective antimicrobial peptides (3000 AU/mL), the planktonic cell numbers of <i>O. oeni</i> decreased from 1.3 x 10¹⁰ cfu/mL to undetectable numbers of viable cells in acidic grape medium. The same experiment performed in modified Chardonnay must yielded no detectable cells of <i>O. oeni.</i>

Antibody-dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores

Using flow cytometry (FCM), an assay has been developed for the determination of antibody-dependent cell-mediated cytotoxicity (ADCC). Target cells were labelled with a membrane dye, PKH-26, to allow discrimination when incubated with effector cells and antibody. Post-incubation, cell death within the PKH-26 + target cell population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3). This ADCC method allows analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the assay is accurate and reproducible with the potential to be a useful tool for evaluating the therapeutic potential of antibodies and antibody-based reagents.

Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide

A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Human T cells express CD86 and acquire APC function after stimulation with human or porcine APC

The interaction between CD86, expressed on antigen presenting cells (APCs), and CD28 on T cells is critical for T cell activation. It has been shown that stimulation of peripheral blood lymphocytes with anti-CD3 and IL-2 induces CD86 expression on T cells, which are capable of providing costimulation to naı̈ve T cells in a human allogeneic system. The goal of our experiments was to determine whether human or porcine APCs can induce CD86 expression on human T cells and whether these T cells can function as APCs. Method: CFSE-labeled human T cells were co-cultured with human or porcine APCs. At day 5, the cultures were harvested and stained with labeled CD86, CD80, CD25 and anti-HLA-DR, CD4, CD8 and TOPRO-3 (viability probe). The samples were analyzed by four-color flow cytometry. Furthermore, T cells were isolated from cultures stimulated with anti-CD3 and Il-2, human APC or Pig APC and these cells were fixed with 1% paraformaldehyde. These cells were used to stimulate fresh syngeneic or allogeneic CFSE-labeled human T cells and these cultures were analyzed at day 7 by flow cytometry. Results: At day 5, cultures stimulated with anti-CD3 and IL-2, allogeneic APCs or porcine APCs show a high percentage (>50%) of proliferating cells expressing CD86 whereas few (2%) non-proliferating cells express CD86. The proliferating cells also expressed CD80, CD25, CD54, Class II and CD45RO. CFSE profiles indicated that fixed T cells expressing CD86 induced by these routes trigger the proliferation of both allogeneic and syngeneic T cells. However, 3H-Thymidine incorporation at day 7 was negative. Conclusion: Human T cells, in response to human or porcine APC stimulation, express CD86 and Class II molecules and function as APCs as assessed by CFSE proliferation assays. Thymidine incorporation data indicates that proliferation had ceased by day 7. This is most likely explained by activation-induced cell death at this time point, since, the proliferating T cells show high levels activation markers CD25 (IL-2R) and CD54 (ICAM-1) and are of memory phenotype (CD45RO positive).

CELL MEDIATED CYTOTOXICITY ASSESSMENT BY RELATIVE CHANGES IN VIABLE TARGET ABSOLUTE COUNTS

We measured absolute counts (cells/μl) of Calcein-AM stained target cells that remained viable [i.e. not permeable to the viability probe 7-AAD (7-Amino Actinomycin D)] following a four-hour incubation with effectors [NK, CTL (cytotoxic T lymphocytes)] without using beads or standards. The absolute counts were evaluated by a Cytoron Absolute (Ortho) flow cytometry apparatus. Median triplicate counts were compared at 0 and 4h, in single targets and increased effector/target ratios. The Coefficient of Variation (CV) of the cell concentration (cells/μl) at the beginning of the experiment was below 6%. The % changes of viable target counts were correlated to the effector/ target ratios by linear regression. The methodology was applied in pairs: 17 for allogenic stem cell transplantation from unrelated donors, 3 of healthy unrelated individuals, 10 for measuring NK activity and 7 autologous. In 15/20 allogenic MLC (mixed lymphocytes culture) and for all NK assays the cytotoxicity was positive (p < 0.05, r2 > 0.9) while in 5/20 allogeneic MLC and in all autologous MLC the outcome was negative (p > 0.05, r2 < 0.4). The proposed method offers the advantage of combining absolute counts with flow cytometry analysis of viable targets, assessment of cell behavior during the cytotoxic phenomenon, the use of a small amount of cells and excellent sensitivity. Our method can estimate frequencies higher than 1:100 for CTL assays.

The acute toxicity of surfactants on fish cells, Daphnia magna and fish—A comparative study

There is a need to replace acute toxicity tests on fish (LC 50) with more cost effective assays. The main objective of this study was to explore whether gill epithelial cells, hepatocytes and Daphnia magna could be used to predict acute toxicity of surfactants on fish. The acute toxicity of 10 synthetic surfactants (anionic, cationic, nonionic and zwitterionic) and two resin acids were determined on hepatocytes and gill epithelial cells from rainbow trout ( Oncorhynchus mykiss), on Daphnia magna and on fish. Cell viability was measured with the fluorescent viability probe calcein–AM, immobilization was determined for Daphnia and 24-hr LC 50 for rainbow trout. The EC 50 values for the cellular tests were clearly higher than the corresponding values for Daphnia and fish, indicating that the cellular tests with the endpoint used are less sensitive than whole organisms. A combination of the EC 50 values for Daphnia and freshly isolated gill epithelial cells in suspension showed, however, a good correlation with acute toxicity on fish (r 2=0.91 and slope=1.09). The combination seems to be a promising in vitro alternative to acute toxicity tests on fish (LC 50), but a more exhaustive comparison, including a broad spectrum of chemicals should be made before the predictive value of the combined in vitro test can be evaluated.

A ruthenium probe for cell viability measurement using flow cytometry, confocal microscopy and time-resolved luminescence.

The capability of the new luminescent probe (dibenzo[h,j] dipyrido[3,2-a:2',3'-c]phenazine)bis(2,2'-bipyridine)ruthenium(II) dication, (RB2Z), to discriminate live and dead cells has been tested on rat hepatocytes and mouse lymphocytes. RB2Z-stained cells were analyzed using flow cytometry, fluorescence (confocal) microscopy and time-resolved luminescence measurements. The established viability probes propidium iodide (PI) and SYTOX green (SG) were used as controls. The intense luminescence of RB2Z at 606 nm is localized in the nucleus of nonviable cells. Viability measurements by flow cytometry and fluorescence microscopy using RB2Z as dead-cell marker yield the same results as PI and SG. The luminescence lifetime of RB2Z also displays sensitivity to cell viability (0.45 and 0.82 microsecond in presence of fully viable and dead cells, respectively). This ruthenium complex is photostable under laser sources and its 200 nm Stokes shift facilitates multicolor labeling experiments in flow cytometry and fluorescence microscopy. Unlike the currently available probes, the long-lived excited state of RB2Z also allows assays based on luminescence decay measurements.

Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry

We report a flow cytometric fluorescence lifetime-based method to discriminate damaged/dead from viable cells in immunofluorescently labeled populations using propidium iodide as a dye-exclusion viability probe. Fluorescence signals from propidium iodide and the anti-thymus cell-surface immunofluorescence marker fluorochromes, phycoerythrin and phycoerythrin/Texas Red (tandem conjugate), which have overlapping emission spectra with propidium iodide, are resolved based on differences in their fluorescence emission lifetimes using phase-sensitive detection. Mouse thymus cell samples were first labeled separately with anti-Thy 1.2 antibody directly conjugated to phycoerythrin and to phycoerythrin/Texas Red and propidium iodide. Labeled cells were then analyzed to determine the lifetimes of the immunofluorescence markers and propidium iodide. Based on these results, rat and mouse thymocytes labeled with anti-Thy 1.1 conjugated to phycoerythrin and anti-Thy 1.2 conjugated to phycoerythrin/Texas Red, respectively, were suspended in phosphate buffered saline containing propidium iodide, and were analyzed as they passed through a flow chamber and crossed a high-frequency, intensity-modulated (sinusoidal) laser excitation beam. The resulting immunofluorescence and propidium iodide signals were resolved based on differences in fluorescence lifetimes expressed as phase shifts using phase-sensitive detection and displayed as frequency distribution histograms and bivariate contour diagrams. This technology provides a new method to resolve immunofluorescence and propidium iodide signals from overlapping fluorescence emission spectra and a flow cytometric lifetime-based technique to quantify damaged/dead cells in immunofluorescence studies.

Gill epithelial cells as tools for toxicity screening—comparison between primary cultures, cells in suspension and epithelia on filters

In the search for suitable cell models that could be used in a test battery for toxicity screening purposes, we have focused on gill epithelial cells from rainbow trout. Gill epithelial cells are attractive models, since the gill is the primary target and uptake site for many toxicants in the water (Evans, D.H., 1987. Environ. Health Perspect. 71, 54–58). The aim of this study was to compare the sensitivity of gill epithelial cells in different test systems: suspensions, primary cultures and epithelia on filters in order to find out which system would be most suitable in a test battery for toxicity screening. The toxicity of the first 30 reference chemicals from the MEIC (multicenter evaluation of in vitro cytotoxicity) project (Bondesson, I., Ekwall, B., Hellberg, S. et al., 1989. Cell Biol. Toxicol. 5, 331–347) to gill epithelial cells in primary culture was tested using the fluorescent viability probe calcein-AM. Ten of the chemicals were also tested on cells suspended after 6–8 days in culture and five of the chemicals were tested on cells cultured on permeable filters. Decrease in transepithelial resistance was used as the endpoint for cells cultured on filters. The results were compared with toxicity data previously obtained by using freshly isolated gill epithelial cells in suspension. There was no significant difference in sensitivity of gill epithelial cells in suspension, primary cultures or resuspended cells. This observation strongly supports the use of cells in suspension in a test battery for toxicity screening purposes. Cultured epithelia on filters proved to be less suitable for screening purposes due to the considerable variation in resistance between epithelia even from the same fish.

Gel immobilization improves survival of Escherichia coli under temperature stress in nutrient-poor natural water

Viable Escherichia coli cells were entrapped in agar gel layers to form artificial biofilms. Sessile-like bacteria and planktonic (suspended) counterparts were exposed for 28 d to natural (spring)water at two temperatures (4°C and 18°C). Culturabilities on non-selective and selective solid media were monitored over the exposure period, together with cell viability that was determined by using a fluorescent viability probe and confocal scanning laser microscopy analysis. The number of planktonic organisms recovered on non-selective medium decreased by 3 logarithmic units during exposure at 4°C and was reduced to an undetectable level at 18°C. Whatever the temperature of the spring water microcosm, however, the immobilized-cell population did not decrease by more than one log unit over the exposure period. Cell counts on coliform-selective, lactose–deoxycholate agar plates confirmed the enhanced resistance of gel-entrapped cells to environment stress as compared to planktonic organisms: at the two tested temperatures, sublethal injury of suspended E. coli reached more than 80% whereas it did not exceed 65% for immobilized cells. Viability studies indicated that planktonic cells rapidly died. These results show that the ability of microorganisms to colonize aquatic ecosystems in the fixed state (i.e. as biofilms) must be considered in studies evaluating cell survival in these environments.

APO2.7 defines a shared apoptotic-necrotic pathway in a breast tumor hypoxia model.

A breast tumor hypoxia model used to simulate conditions which may exist within an enlarging tumor was examined using documented methods for identifying mechanisms of cell death and compared to the mitochondrial membrane-specific APO2.7 antigen expression. Hypoxic conditions were induced by holding cell pellets of MDA-MB-175-VII breast carcinoma cells in tightly capped centrifuge tubes for up to 10 days. Cells were harvested at 1.5, 3, 4.5, 6, 12, 18, and 24 h, and each 24 h thereafter to 10 days. APO2.7 was monitored in unprocessed cells (no permeabilization prior to staining) for all time points and processed cells (permeabilized prior to staining) for only the first 24 h. Cell viability probes trypan blue and anti-tubulin antibody showed a rapid increase in staining over the first 24 h, as did the phosphatidylserine-specific annexin V and DNA fragmentation by flow cytometry (range of 60-81% positive staining). Light scatter changes indicative of cell death were also quite remarkable. APO2.7 staining never exceeded 42% of the cell pellet over the 10 days of testing compared to greater than 95% staining for all other methods tested. When APO2.7 antigen expression was examined with respect to depth in the cell pellet, it was apparent that cells deeper in the pellet expressed APO2.7 more rapidly; however, fewer cells stained and cells showed fewer apoptotic features on an ultrastructural level than cells at the cell media interface. The study indicates that the anti-APO2.7 antibody may be able to discern apoptotic and incomplete apoptotic cells from necrotic MDA-MB breast cancer cells, traversing a heterogeneous pathway to cell death induced by hypoxia.

Bacteria in post-glacial freshwater sediments.

Prokaryote communities in post-glacial profundal freshwater sediments of Windermere, representing 10-12,000 years of deposition, were examined for culturability, viability and community structure. The potential for active geochemical cycles was inferred from the presence of specific groups of bacteria. Direct count procedures revealed 10(12) cells (g dry wt sediment)-1 in the surface sediments, which declined to approximately 10(9) cells (g dry wt sediment)-1 at 6 m depth of core (Representing approximately 10,000 years of deposition). The majority of the cells in the upper sediments were metabolically active when challenged with viability probes and responded to the direct viable count method. Below 250 cm, viability shown by 5-cyano-2,3-diotyl tetrazolium chloride (CTC) dye was not significantly different from the direct count; however, counts obtained with 5-carboxyfluorescein diacetate (CFDA) and the direct viable count both declined significantly from the direct count below 250 cm and 1 m, respectively. Culture was achieved from samples throughout the core, although the numbers of culturable bacteria decreased significantly with depth, from 10(7) c.f.u. (g dry wt sediment)-1 to 10(1)-10(2) c.f.u. (g dry wt sediment)-1 below 3 m depth. Among culturable isolates, Gram-positives and Gram-negatives were found at all levels of the core, and spore-forming heterotrophs dominated. Although sulphate-reducing bacteria were not detected below 20 cm, isolates demonstrating denitrifying activity were detected at all depths. PCR performed on samples taken below 3 m (deposited more than 7000 years ago) using eubacterial and archaeal primers revealed sequences similar to those found in deep sediments of the Pacific Ocean and the presence of methanogenic archaea. These observations indicate that bacteria and archaea are capable of long-term persistence and activity in deep, aged freshwater sediments.

Substratum topography influences susceptibility of Salmonella enteritidis biofilms to trisodium phosphate.

Established (48- and 72-h) Salmonella enteritidis biofilms grown in glass flow cells with or without artificial crevices (0.5-, 0.3-, and 0.15-mm widths) were subjected to a 10% trisodium phosphate (TSP) solution under different flow regimens (0.3, 0.6, 1.2, and 1.8 cm s-1). The abundance of biofilm remaining after TSP treatment, the biocidal efficacy of TSP, and the factors which contributed to bacterial survival were then evaluated by using confocal laser microscopy and a fluorescent viability probe. Biofilm age affected the amount of biofilm which remained following a 15-s exposure to TSP. After TSP treatment of 48-h biofilms, 29% of the original biofilm remained at the biofilm-liquid interface, whereas 75% of the biofilm remained at the base (the attachment surface). Following TSP treatment of 72-h biofilms, 27% of the biofilm material remained at the biofilm-liquid interface, 73% remained at the 5-micron depth, and 91% remained at the biofilm base. Results obtained using the BacLight viability probe indicated that TSP exposure killed all the cells in 48-h biofilms, whereas in the thicker 72-h biofilms, surviving bacteria (approximately 2% of the total) were found near the 5- and 0-micron depths. In the presence of artificially constructed crevices, an inverse relationship was shown to exist between bacterial survival (ranging from approximately 13 to 83% of total biofilm material) and crevice width. This relationship was further influenced by the velocity of TSP flow; high TSP flow velocities (1.8 cm s-1) resulted in the lowest number of surviving bacteria at the base of crevices (approximately 42% survival). Extended time courses demonstrated that after TSP stress was relieved, biofilms continued to grow within crevices but not in systems without crevices. It is suggested that advective TSP flux into crevices and through the biofilm matrix was enhanced under conditions of high flow. These results suggest that the inherent roughness of the substratum on which the biofilm was grown and the timing of TSP application are important factors controlling the efficacy of TSP treatment.

Fluorescent probes for wall porosity and membrane integrity in filamentous fungi

To assess the viability of moulds, quick detection methods using fluorescent probes were evaluated. A major hurdle in the use of intracellular probes can be the wall of filamentous fungi. We studied the permeability of the wall of germinating conidiospores with anionic ellipsoid FITC-dextran molecules and confocal scanning laser microscopy. In contrast to S. cerevisiae, filamentous fungi internalized FITC-dextran molecules up to 150 kDa (Stokes radius of 8–9 nm). We concluded that the wall of germinating conidia forms no size exclusion barrier for fluorescent viability probes. To assess the viability of a suspension of germinating conidiospores, PI, CDFDA, and FUN1 were shown to be suitable. It was not possible to homogeneously stain fresh conidiospores with fluorescent viability probes. Nonetheless, fluorescence activated cell sorting was possible and allowed the sorting of different sub-populations. Whilst the presence of esterase activity (CDFDA staining) did not correlate with outgrowth potential, the uptake of PI did correlate with no outgrowth.

The use of freshly isolated gill epithelial cells in toxicity testing

The large number of chemicals in our environment, many of which have been insufficiently tested for toxicity, justifies the need for rapid and reliable in vitro testing to predict toxicity. In aquatic toxicology the use of cellular toxicity tests has been limited. In the present study, the usefulness of freshly isolated gill epithelial cells from rainbow trout in toxicity testing was evaluated by testing the cytotoxicity of 30 reference chemicals to the cells using the fluorescent cell viability probe Calcein-AM. The results were compared with previously obtained EC 50 values for the chemicals in freshly isolated rainbow trout hepatocytes, EC 50 values for immobilization of Daphnia magna and literature data on LC 50 values for fish. Regression analysis of the data obtained with gill epithelial cells and hepatocytes showed a correlation coefficient of 0.77 (r 2 = 0.59). The gill epithelial cells were slightly more sensitive than hepatocytes to the chemicals. The slope of the regression line was 0.79. Both cell types were less sensitive than D. magna and fish to the chemicals, but the EC 50 values for gill epithelial cells showed a better concordance with EC 50 values for immobilization in D. magna and with literature data for fish LC 50 values than the hepatocyte EC 50 values.

Keratocyte networks visualised in the living cornea using vital dyes.

Fluorescent viability probes have been used to visualise and investigate the viability, morphology and organisation of the keratocyte within the stroma of the intact living cornea. The live cell probe, calcien-AM, in combination with a dead cell probe, ethidium homodimer (Live/Dead Assay, Molecular Probes, U.S.A.) proved superior to earlier generation vital dyes such as fluorescein diacetate or 5,6-carboxyfluorescein diacetate, initially used in combination with ethidium bromide. The ubiquitous distribution of esterase enzymes that cleave calcien-AM within the keratocyte cytoplasm produced a high concentration of fluorescently active calcein throughout the cell, including fine cell processes. Epi-illuminated fluorescence microscopy on transparent corneal dissections subsequently revealed details of keratocyte microanatomy and three-dimensional network organisation in situ. Three morphologically discrete subpopulations of keratocytes were identified: two formed relatively small bands of cells, immediately subjacent to either Bowman's or Descemet's membranes, the third subpopulation constituting the majority of keratocytes typically located within the corneal stroma. The results indicate that calcein-AM is able to penetrate intact living cornea revealing cell viability, and it also has the capacity to 'trace' cellular elements and reveal fine structure within a dense connective tissue matrix.