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PubMed1987

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.

Biodegradation, Environmental↗

PubMed1982

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts.

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking-focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.

Actins↗

PubMed1995

Response of human gingival fibroblasts to prostaglandins.

The effects of various prostaglandins (PGs) on the functions of human gingival fibroblasts (Gin-1 cells; ATCC CRL 1292) were examined by phase-contrast microscopy, cell-counting and radioautographic experiments. Tested PGs were PGA1, PGA2, PGB1, PGB2, PGD2, PGE1, PGE2, PGF1 alpha, PGF2 alpha, PGI2, 6-keto-PGF1 alpha, 9 alpha-11 alpha-methanoepoxy-PGF2 alpha, and thromboxane (TX) B2. PGA1 and PGD2 at 30 microM caused morphological deformation of Gin-1 cells. All the PGs tested at 30 microM suppressed the proliferation of Gin-1 cells in the logarithmic growth phase. Furthermore, all the PGs tested at 10 microM suppressed DNA synthesis, collagen synthesis, and noncollagenous protein synthesis in confluent Gin-1 cells, while exerting no effect on GAG synthesis. The concentrations of PGs used are beyond those found in healthy gingiva. However, in periodontitis the local concentrations of some PGs within the gingiva are expected to be extremely elevated beyond the physiological level. These results suggest that PGs may play an important role as a negative regulator in metabolism and some pathologic gingival conditions by suppressing the functions of gingival fibroblasts.

Cell Division↗

PubMed2002

Inhibition of mouse in vitro fertilization by an antibody against a unique 18-amino acid domain in the polysulfate-binding domain of proacrosin/acrosin.

OBJECTIVE: To determine the contribution of the polysulfate-binding domain (PSBD) of acrosin during sperm penetration. DESIGN: To inhibit the in vitro fertilization of mouse zona-intact oocytes by using a polyclonal antibody raised against an 18-amino acid peptide of proacrosin (anti-PSBD). SETTING: Unit of Reproduction and Development, Faculty of Biological Sciences, Pontifical Catholic University of Chile. PATIENT(S): None. INTERVENTION(S): A polyclonal antibody against the 43IFMYHNNRRYHTCGGILL(60) peptide was raised in New Zealand female rabbits. The specificity of the antibody was evaluated by an ELISA. Zona-intact mouse oocytes were coincubated with capacitated spermatozoa for 3 hours in the presence of 0.63 mg/mL of the antibody or preimmune serum. As a control, we used zona-free mouse oocytes under the same experimental conditions. MAIN OUTCOME MEASURE(S): We evaluated the fertilization rate of zona-intact and zona-free mouse oocytes by phase-contrast microscopy. An oocyte was considered fertilized when at least one decondensed sperm head was found within the egg cytoplasm. We evaluated 50-60 mouse oocytes in each group in three independent experiments. RESULT(S): The anti-PSBD antibody inhibited the fertilization of zona-intact, but not zona-free, mouse oocytes, by capacitated spermatozoa. In addition, the binding of the anti-PSBD to proacrosin/acrosin in a solid-phase assay was inhibited in the presence of polysulfates (fucoidan). CONCLUSION(S): The anti-PSBD directed against the PSBD of proacrosin/acrosin inhibited the penetration of capacitated mouse spermatozoa through the zona pellucida. This antibody may be a useful tool to define the roles of the different domains of proacrosin/acrosin during gamete interaction.

Acrosin↗

PubMedDate not supplied

Human HT-29 colon carcinoma cells: mucin production and tumorigenicity in relation to growth phases.

The main aim of this study was to determine whether changes in mucin production/secretion during the growth phases of a human adenocarcinoma cell line, HT-29, are associated with a different tumorigenic potential. HT-29 cells cultured in DMEM supplemented with 10% FCS were harvested in the exponential and post-confluent phases of growth. Metabolic labeling of the cells was performed using [3H]-glucosamine. Following a 24 hr-incubation period, radioactivity was measured in 1 ml-aliquots of cell cytosol and culture medium. Concurrently, mucin synthesis was assessed by size exclusion chromatography of [3H]-glucosamine-labeled glycoprotein in a Sepharose CL-4B column. Clonigenic assay in soft agar of pre- and post-confluent HT-29 cells was determined by scoring viable colonies according to size and number using phase-contrast microscopy. To assess in vivo tumorigenicity, pre-and post-confluent HT-29 cells (4 x 10(6)) in 0.2 ml DMEM were inoculated into nude mice. Tumor size and volume were recorded for 31 days. H-29 cells grew as multilayers of unpolarized, undifferentiated cells. Colony forming efficiency was similar at all growth stages. A significant increase in mucin synthesis was noted in HT-29 cells harvested in the exponential phase of growth compared to the stationary phase (148.3 +/- 41.9 versus 49.1 +/- 5.0, mean +/- SE, dpm/4 x 10(3) cells, p < 0.05). Mucin secretion was not significantly changed. Tumorigenicity in nude mice was consistently higher when the cells were injected in the exponential phase of growth. On day 31 after cell inoculation the average tumor volume/site was 332.7 mm3 in mice injected with HT-29 cells in log phase compared to 142.7 mm3 (p < 0.01) in animals which received post-confluent cells. Tumor weights were 0.36 g and 0.22 g respectively (p < 0.05). The present results indicate a definitive correlation between growth phases of HT-29 cells and mucin synthesis: mucin production was significantly higher in exponentially growing cells. The cloning efficiency in soft agar, a marker of in vitro tumorigenicity, was similar in HT-29 cells irrespective of the growth stage. A main finding of the present study was the increased in vivo tumorigenicity of colon cancer cells inoculated into athymic mice in the log phase of growth compared to cells harvested at post-confluence. These results are consistent with the view that mucin plays an important contributory role in determining tumorigenicity.

Animals↗

PubMed1998

Quantitative assessment of leading edge adhesion: reattachment kinetics modulated by injury-derived intracellular calcium predict wound closure rates in endothelial monolayers.

Migrating cells continually develop new substrate attachments at the leading edge (LE) in order to maintain traction for movement. This study evaluates the relationship between LE adhesion and wound closure by modulating injury-derived intracellular free Ca2+ ([Ca2+]i) signaling in endothelial cell (EC) monolayers following scrape-wounding. These data show that brief treatment with increased extracellular Ca2+ ([Ca2+]e) during wounding accelerated wound area closure rates by 50-65%, while brief treatments with calcium influx inhibitors reduced rates by 30-50%. Fura-2 studies in wounded monolayers indicated supranormal [Ca2+]e during wounding increased (by 52%), while influx-inhibitors decreased (by 36%) the percentage of cells exhibiting elevated plateau [Ca2+]i levels. Quantitative time-lapse interference reflection microscopy (IRM) together with indirect alphavbeta3 integrin immunofluorescence was used to measure the effects of 100 microM Gd3+ and 5 mM [Ca2+]e treatment on fractional LE adhesion after wounding. Influx inhibition blocked development of increased injury-derived LE adhesion. Measurements indicated a linear relationship (r2 = 0.99, 0.98) between LE adhesion, development rates (quantified as an association rate constant) and steady state wound closure rates. Changes in filopodial activity, as indicated by phase contrast microscopy, did not correlate with changes in wound closure rates, but an association existed between the percentile peak [Ca2+]i response and the initiation of filopodial activity, suggesting a role for filopodia in mediating Ca2+-sensitive acceleration. Taken together, our data suggest that injury-derived [Ca2+]i signaling may regulate wound closure rates by an adhesion-mediated mechanism.

Animals↗

PubMed1994

Staurosporine induces the outgrowth of neurites from the dorsal root ganglion of the chick embryo and PC12D cells.

Staurosporine, a potent inhibitor of protein kinases, caused the rapid outgrowth of neurites from cultured dorsal root ganglia of chick embryos and from PC12D cells, a subline of PC12 cells. Treatment of dorsal root ganglia with 1 to 20 nM staurosporine resulted in the extensive outgrowth of neurites that were indistinguishable from those induced by NGF, as assessed by phase-contrast microscopy, electron microscopy and cytochemical staining of actin and tubulin. However, neurites generated from the ganglia in response to the higher concentrations of staurosporine (40-100 nM) seemed to have different characteristics, possibly as a result of the inhibition of cell migration from ganglia. The sequential changes in morphology of PC12D cells in response to staurosporine and to NGF were revealed by staining of actin. Ruffling membranes emerged at the margins of PC12D cells within 4 min after the addition of staurosporine or of NGF. From 10 min to 24 h after the addition of either compound, the ruffles were transformed into several projections that became growing neurites. The formation of ruffles and the outgrowth of neurites were both apparent at a concentration of staurosporine of 10 nM. The neurites that emerged from PC12D cells in response to staurosporine and in response to NGF were indistinguishable under the phase-contrast microscope and after staining of actin and tubulin. However, staurosporine never promoted survival of PC12D cells in serum-free conditions as that promoted by NGF. The observations indicate that staurosporine at nanomolar concentrations may reproduce the neurogenic changes that induced by NGF in primed neuronal cells, although it can not mimic the action of NGF that supports survival of neurons.

Alkaloids↗

PubMed1982

Monolayer culture of human endometrium: methods of culture and identification of cell types.

Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture.

Cells, Cultured↗

PubMed1972

Improved staining of extracellular polymer for electron microscopy: examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus.

Phase contrast, ultraviolet microscopy, and freeze-etching were used to determine the amount of exocellular polymer surrounding unfixed cells of four genera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mesenteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectional electron microscopy was employed to measure the effectiveness of a modified ruthenium red staining method. The results obtained with this modification of ruthenium red staining technique were compared to results obtained when previously proposed ruthenium red methods of fixation were employed. The results of these relations were then compared to the amounts of exocellular material as determined with phase-contrast microscopy, ultraviolet microscopy, and freeze-etching. The data obtained indicate that improved fixation of exocellular polymer is achieved when cells are pretreated with ruthenium red as described herein. In addition, the modified methods also reveal cytological detail not apparent when other methods of ruthenium fixation are employed.

Azotobacter↗

PubMed1999

Timing of FtsZ assembly in Escherichia coli.

The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

Animals↗

PubMed1991

Cytosolic Ca2+ deregulation and blebbing after HgCl2 injury to cultured rabbit proximal tubule cells as determined by digital imaging microscopy.

Acute injury to renal proximal tubule cells has previously been shown to result in elevated cytosolic Ca2+ ([Ca2+]i), blebbing, and eventual cell death. In this study, digital imaging fluorescence microscopy was used to evaluate these changes in response to HgCl2 treatment of cultured rabbit proximal tubular cells. Monolayer cells loaded with fura-2 were treated with 10, 50, or 100 microM HgCl2 in both 1.37 mM CaCl2-containing and nominally Ca(2+)-free (less than 5 microM) Hanks' balanced salt solution. [Ca2+]i was estimated by measuring the ratio of fluorescent image pairs (collected at 340- and 380-nm excitation), morphological changes were observed by phase-contrast microscopy, and viability was assessed by trypan blue exclusion. After exposure of cells to 10 microM HgCl2, [Ca2+]i initially increased about 2-fold by 5 min; after 50 or 100 microM HgCl2, [Ca2+]i rapidly rose 2- to 3-fold, peaked at 1-3 min, and then generally decreased slightly. In nominally Ca(2+)-free (less than 5 microM) medium, [Ca2+]i stabilized, but in 1.37 mM Ca(2+)-containing medium, [Ca2+]i continued to slowly rise, often reaching levels of fura-2 saturation. The rate and extent of blebbing and the rate of cell death were increased in the presence of 1.37 mM Ca2+. These results show that sustained elevations of [Ca2+]i precede both cell blebbing and cell death and that when these elevations are limited by removing extracellular Ca2+ the amount of blebbing is reduced and cell viability is prolonged.

Animals↗

PubMed1987

Electron microscopical studies of Strongyloides ratti infective larvae: loss of the surface coat during skin penetration.

Previous indications using radiolabelled larvae that Strongyloides ratti free-living infective larvae lose a surface coat during penetration of the skin were further investigated by transmission electron microscopy of the cuticle of S. ratti infective larvae in the free-living stage, after penetration of mouse skin, and after migration to the lungs. These studies demonstrated the presence of a faint electron-dense surface coat external to the epicuticle on free-living worms which was absent from larvae recovered from the skin and lungs. When free-living infective larvae were incubated in 10% CO2 at 37 C and then examined with phase-contrast microscopy, worms were observed in the process of losing this coat. These observations confirm the hypothesis that S. ratti infective larvae lose a surface coat during penetration of the skin.

Animals↗

PubMed1995

In vitro evaluation of donor liver preservation fluids on human hepatocyte function.

Successful liver transplantation depends on adequate preservation of cellular function. We therefore tested the effects of two currently used liver preservation fluids, Euro-Collins (EC) solution and University of Wisconsin (UW) solution, on the viability and some functional activities of hepatocytes isolated from human livers. Cells in primary culture were maintained under hypoxic (95% N2/5% CO2) and hypothermic (4 degrees C) conditions for 24 h, either in EC or UW solution. This treatment did not result in significant hepatocyte damage, as judged by phase contrast microscopy, intracellular LDH release, and the MTT mitochondrial test. However, neutral red uptake indicated that lysosomal functions were slightly affected (35% decrease) when compared to control conditions. At the end of the hypoxia/hypothermia period, hepatocyte monolayers were incubated at 37 degrees C under normoxic conditions for 24 h, in order to simulate the reperfusion of a transplanted liver. Three drugs--midazolam, diazepam, zidovudine--were used as diagnostic substrates to check the metabolic abilities of human hepatocytes replaced in normal conditions. Both phase I (hydroxylation, demethylation) and phase II (glucuronidation) metabolic reactions were affected by the hypoxia/hypothermia shock. Indeed, a 30%-50% decrease in these activities was observed as compared to values obtained in control hepatocytes. No difference could, however, be found at the cellular level regarding the solution used for cold storage. These results suggest that the superiority of UW over EC solution, already reported in clinical practice after transplantation of preserved human livers, was not due to a better preservation of the hepatocytes.

Antimetabolites↗

PubMed1988

Evidence for sequential deployment of secretory enzymes during the normal acrosome reaction of guinea pig sperm in vitro.

Experiments were conducted to determine if acrosomal enzymes are released simultaneously or in sequence during the normal acrosome reaction. Epididymal guinea pig sperm were incubated in a chemically defined, calcium-containing medium which supports normal acrosome reactions within 4-5 hours at 37 degrees C. The sperm suspensions were monitored for motility, normal acrosome reactions, and false acrosome reactions during in vitro incubation. At specified time intervals, the sperm were separated from the incubation medium by centrifugation, and the distribution of dipeptidyl peptidase (DPP II) and acrosin activity was determined by biochemically assaying the hydrolysis of trialanine and N-benzoyl-L-arginine ethyl ester (BAEE), respectively. When calcium was present, there was a significant increase in DPP II activity in the supernatants by 1 hour of incubation and a slight decline at later time points. This release was not correlated with false or normal acrosome reactions (loss of the acrosomal cap) monitored by phase-contrast microscopy but probably represents a very early stage in the normal acrosome reaction. This early stage is difficult to detect at the light microscope level because sperm are still in rouleaux and because membrane fusion is not directly observable. In contrast, acrosin activity, which was assayed in the same supernatants, increased at later times when sperm were observed to have completed normal acrosome reactions. The ultrastructural distribution of DPP II was determined in sperm pellets collected during in vitro incubation by using the DPP II substrate lysyl-alanyl-4-methoxy-2-naphthyamide. In freshly isolated cauda epidiymal sperm, reaction product is confined to the light-staining area in the dorsal bulge of the acrosome. However, by 1 hour of incubation, the light-staining area of many sperm was partially or completely dispersed, while other regions of the acrosome were unchanged. Our data are consistent with the conclusions that DPP II is a highly soluble component of the guinea pig sperm acrosome and that its release occurs during the initial phase of the acrosome reaction while sperm are still in rouleaux. Structural changes in the acrosome associated with DPP II release were detectable by electron microscopy but not by light microscopy. Acrosin, which is less soluble than DPP II, is released at a later time during the acrosome reaction. Both DPP II and acrosin appear to be partially inhibited following their release from sperm. A complete understanding of the sequential release and extracellular activities of the acrosomal enzymes will be necessary to fully define their functions in fertilization.

Acrosin↗

PubMed2003

Image analysis of tendon helical superstructure using interference and polarized light microscopy.

Wave-like structures (WLS also known as crimp) have generally been reported to be planar structures. However, there is evidence that a helical superstructure, rather than a planar one, should be considered. Conditions dictated by supramolecular chemistry, molecular recognition and self-assembly favor the idea of a helical arrangement for collagen bundles in a supramolecular structure. The aim of this work is to provide additional data in support of a helical superstructure for collagen bundles in tendons. Cryosections of fixed flexor bovine tendons and sections of resin-embedded peeled rat tail were studied using polarized light, interference, and phase contrast microscopy. Image analysis was used to find appropriate mathematical descriptors for WLS. Interference colors due to the dispersion of birefringence allowed the detection of a gradual, intertwined twisted fiber organization in WLS, as the angle of the tendon axis was rotated relative to the polarizers. Helical movements of the images of the WLS bands were produced using animation methods. Interference microscopy revealed interference colors associated with different orientations and dry mass concentrations in the fibers, especially in tendon cross-sections, which also exhibited Maltese-cross birefringence images. Similar images were detected by interference microscopy, suggesting a spiral organization of fibers in the section plane. The helical orientation of the fibers was detected by focusing through different planes of sections. Based on a comparison of this superstructure with mesophases, the twisted grain boundary concept is considered to be the most appropriate for the classification of tendon WLS.

Animals↗

PubMed1999

Direct or referral microscopy of vaginal wet smear for bacterial vaginosis: experience from an STD clinic.

A new wet smear diagnostic criterion for bacterial vaginosis was applied to 124 consecutive female patients attending an STD clinic located in the centre of Copenhagen. Bacterial vaginosis was detected in 54 (44%) women, making bacterial vaginosis the most prevalent pathological condition encountered. A total of 47 (87%) of the women were symptomatic. Concomitant genital infections were found in 13 (24%) of these women, most often as vulvo-vaginal candidiasis. A correct microscopic diagnosis could also be obtained by sending the vaginal smear to the local microbiologist for rehydration and phase contrast microscopy. It is suggested that the previously described vaginal wet smear criteria are used in place of Amsel's criteria for routine diagnosis of bacterial vaginosis.

Adult↗

PubMed1992

Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.

A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.

Animals↗

PubMed2000

Robust autofocusing in microscopy.

BACKGROUND: A critical step in automatic microscopy is focusing. This report describes a robust and fast autofocus approach useful for a wide range of microscopic modalities and preparations. METHODS: The focus curve is measured over the complete focal range, reducing the chance that the best focus position is determined by dust or optical artifacts. Convolution with the derivative of a Gaussian smoothing function reduces the effect of noise on the focus curve. The influence of mechanical tolerance is accounted for. RESULTS: The method is shown to be robust in fluorescence, bright-field and phase contrast microscopy, in fixed and living cells, as well as in fixed tissue. The algorithm was able to focus accurately within 2 or 3 s, even under extremely noisy and low contrast imaging conditions. CONCLUSIONS: The proposed method is generally applicable in light microscopy, whenever the image information content is sufficient. The reliability of the autofocus method allows for unattended operation on a large scale.

Animals↗