Confocal Laser Scanning Microscopy Data Research Articles

A two-step procedure for automatic and accurate segmentation of volumetric CLSM biofilm images

This paper presents a robust two-step segmentation procedure for the study of biofilm structure. Without user intervention, the procedure segments volumetric biofilm images generated by a confocal laser scanning microscopy (CLSM). This automated procedure implements an anisotropic diffusion filter as a preprocessing step and a 3D extension of the Otsu method for thresholding. Applying the anisotropic diffusion filter to even low-contrast CLSM images significantly improves the segmentation obtained with the 3D Otsu method. A comparison of the results for several CLSM data sets demonstrated that the accuracy of this procedure, unlike that of the objective threshold selection algorithm (OTS), is not affected by biofilm coverage levels and thus fills an important gap in developing a robust and objective segmenting procedure. The effectiveness of the present segmentation procedure is shown for CLSM images containing different bacterial strains. The image saturation handling capability of this procedure relaxes the constraints on user-selected gain and intensity settings of a CLSM. Therefore, this two-step procedure provides an automatic and accurate segmentation of biofilms that is independent of biofilm coverage levels and, in turn, lays a solid foundation for achieving objective analysis of biofilm structural parameters.

Morphology, mechanical properties and viability of encapsulated cells

The morphological and mechanical properties of encapsulated yeast cells ( Saccharomyces cerevisiae) have been investigated by atomic force microscopy (AFM). Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte (PE) layers. The properties of cells coated by different numbers of PE layers and from PE solutions of different ionic strength have been investigated. AFM imaging indicates an increase in PE coating stability when decreasing the solution ionic strength. The Young's moduli of the different examined systems have been evaluated through a quantitative analysis of force–distance curves by using the Hertz–Sneddon model. The analysis indicates an increase in hybrid system stiffness when lowering the ionic strength of the PE solution. An evaluation of the viability of encapsulated cells was obtained by confocal laser scanning microscopy (CLSM) measurements. CLSM analysis indicates that cells preserve their subcellular structure and duplication capability after encapsulation. By coupling AFM and CLSM data, a correlation between local stiffness and duplication rate was obtained.

Fluorescent Bacterial Magnetic Nanoparticles as Bimodal Contrast Agents

The purpose of this study was to assess whether fluorochrome-coupled bacterial magnetic nanoparticles can be used as bimodal contrast agent for both magnetic resonance imaging (MRI) and near-infrared fluorescence optical (NIRF) imaging of cultured macrophages. Bacterial magnetic nanoparticles (magnetosomes, particle diameter: 42 nm) were harvested from Magnetospirillum gryphiswaldense and characterized by using MRI. After covalent coupling to the fluorescent dye DY-676 (lambdaabs./lambdaem.= 676 nm/701 nm, Dyomics, Jena, Germany), the fluorescent magnetosomes were analyzed by fluorescence-activated cell sorting. Subsequently, murine macrophages J774 were incubated with the bimodal contrast agent (3 hours) and examined by a whole-body near infrared small animal imaging system as well as by using a 1.5 T clinical MR system. Moreover, labeled cells were characterized using confocal laser scanning microscopy (CLSM) and ultrathin section transmission electron microscopy. Characterization of the nanoparticles by MRI revealed R1 and R2 relaxivities of 3.2 mMs and 526 mMs, respectively. Fluorochrome-coupled magnetosomes exhibited increased fluorescence intensities at wavelengths >670 nm. Macrophages that were incubated with the contrast agent showed a significant fluorescence emission in the near infrared range as imaged with a whole body NIR imaging system, FACS analysis and CLSM. Moreover, CLSM data showed the greatest fluorescence intensities within intracellular compartments and colocalized with the magnetosomes. With MRI, both T1 and T2 relaxation times were substantially shortened at concentrations greater than 600 cells/microL. Macrophages could be labeled with fluorescent magnetosomes, and they were successfully imaged using both a 1.5 T MR scanner as well as with NIRF optical methods. The use of this bimodal contrast agent for diagnostic purposes may benefit from the excellent spatial resolution of the MRI and the high sensitivity of the fluorescence imaging.

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device.

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD – an apparatus that was designed for high-throughput susceptibility testing – allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.

Effect of membrane fouling reducer on cake structure and membrane permeability in membrane bioreactor

The nature of the cake formed on a membrane surface is a key factor for understanding membrane fouling phenomenon in membrane bioreactor (MBR) systems. Most studies on membrane fouling have been carried out with the analysis of microbial characteristics in the bulk phase rather than in the cake because the preparation of intact cake specimen is hardly possible. In this study, the cake on a membrane surface in a MBR system was non-destructively examined using confocal laser scanning microscopy (CLSM) and digital image analysis. This CLSM-Image analysis technique was then applied to elucidate the mechanism of significant flux enhancement that occurs when a membrane fouling reducer (MFR) was added to MBR. Even though the cake of the MFR reactor was thicker than that of the control reactor, membrane fouling was decreased primarily as the result of increased cake porosity. The concentration of soluble extra-cellular polymeric substances (EPS) in the MFR reactor was lower, but the bound EPS concentration was higher than that in the control reactor. Cakes formed in the presence vis-à-vis at the absence of MFR were compared in terms of structural features and the spatial distribution of cellular and polymeric cake constituents which were determined using the CLSM data and image analysis.

Modeling and simulation of protein uptake in cation exchanger visualized by confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) has been extensively applied in the area of protein chromatography to investigate the uptake mechanism of protein in adsorbents. However, due to the light attenuation in the deeper layers of a specimen, quantitative analysis using CLSM data is still far from reality. In this work, an attenuation equation for describing the darkening of the CLSM image in the deeper scanning layers was developed. Bovine serum albumin (BSA) adsorption to SP Sepharose FF was performed by batch adsorption and micro-column chromatography on which protein concentration in single absorbents were visualized by CLSM. The parameters in the equation were estimated by fitting it to the fluorescence intensity profiles obtained at adsorption equilibrium, and then the equation was used to simulate the effect caused by the light scattering and absorption. CLSM analysis demonstrated that BSA adsorption to SP Sepharose FF followed the shrinking core pattern and was predicted reasonably well by the pore diffusion model in combination with the attenuation equation. By comparison of the CLSM data with the simulations, it shows that the attenuation equation was useful to demonstrate the validity of an intraparticle mass transport model for the estimation of intraparticle protein concentration profiles.

Application of Phase Correlation to the Montage Synthesis and Three-Dimensional Reconstruction of Large Tissue Volumes from Confocal Laser Scanning Microscopy

We have implemented and tested a new automatic method for the montage synthesis and three-dimensional (3D) reconstruction of large tissue volumes from confocal laser scanning microscopy data (CLSM). This method relies on maximization of the phase correlation between adjacent images. It was tested on a large specimen (a murine heart) that was cut into a number of individual sections with thickness appropriate for CLSM. The sections were scanned horizontally (in-plane) and vertically (perpendicular to the optical planes) to produce "tiles" of a 3D volume. Phase correlation maximization was applied to the montage synthesis of in-plane tiles and 3D alignment of optical slices within a given physical section. The performance of the new method is evaluated.

Novel methodology utilizing confocal laser scanning microscopy for systematic analysis in arthropods (Insecta)

The use of confocal laser scanning microscopy (CLSM) for imaging arthropod structures has the potential to profoundly impact the systematics of this group. Three-dimensional visualization of CLSM data provides high-fidelity, detailed images of minuscule structures unobtainable by traditional methods (for example, hand illustration, bright-field light microscopy, scanning electron microscopy). A CLSM data set consists of a stack of 2-D images ("optical slices") collected from a transparent, fluorescent specimen of suitable thickness. Small arthropod structures are particularly well suited for CLSM imaging owing to the autofluorescent nature of their tissues. Here, we document the practical aspects of a methodology developed for obtaining image stacks via CLSM from autofluorescent insect cuticular structures.

Effects of Feeding Spodoptera littoralis on Lima Bean Leaves. III. Membrane Depolarization and Involvement of Hydrogen Peroxide

In response to herbivore (Spodoptera littoralis) attack, lima bean (Phaseolus lunatus) leaves produced hydrogen peroxide (H(2)O(2)) in concentrations that were higher when compared to mechanically damaged (MD) leaves. Cellular and subcellular localization analyses revealed that H(2)O(2) was mainly localized in MD and herbivore-wounded (HW) zones and spread throughout the veins and tissues. Preferentially, H(2)O(2) was found in cell walls of spongy and mesophyll cells facing intercellular spaces, even though confocal laser scanning microscopy analyses also revealed the presence of H(2)O(2) in mitochondria/peroxisomes. Increased gene and enzyme activations of superoxide dismutase after HW were in agreement with confocal laser scanning microscopy data. After MD, additional application of H(2)O(2) prompted a transient transmembrane potential (V(m)) depolarization, with a V(m) depolarization rate that was higher when compared to HW leaves. In transgenic soybean (Glycine max) suspension cells expressing the Ca(2+)-sensing aequorin system, increasing amounts of added H(2)O(2) correlated with a higher cytosolic calcium ([Ca(2+)](cyt)) concentration. In MD and HW leaves, H(2)O(2) also triggered the increase of [Ca(2+)](cyt), but MD-elicited [Ca(2+)](cyt) increase was more pronounced when compared to HW leaves after addition of exogenous H(2)O(2). The results clearly indicate that V(m) depolarization caused by HW makes the membrane potential more positive and reduces the ability of lima bean leaves to react to signaling molecules.

Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.

Oxidative quenching of spruce thermomechanical pulp fiber autofluorescence monitored in real time by confocal laser scanning microscopy-implications for lignin autofluorescence.

Confocal laser scanning microscopy (CLSM) was used to monitor real-time lignin autofluorescence intensities from different cell wall compartments of spruce fibers during oxidation by laccase-2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate (ABTS) treatment. CLSM data revealed an instant emission quenching from all cell wall compartments, including those not physically accessible to enzyme or ABTS, followed by an additional decrease simultaneously in all cell wall compartments over a 45 min time course. The importance of site-to-site excitation energy transfer in lignin efficient over long distance is suggested.

Quantitative imaging of cis-regulatory reporters in living embryos.

A confocal laser scanning microscopy method has been developed for the quantitation of green fluorescent protein (GFP) as a reporter of gene activity in living three-dimensional structures such as sea urchin and starfish embryos. This method is between 2 and 50 times more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo. By using coinjected Texas red dextran as an internal fluorescent standard, the observed GFP intensity is corrected for variations in laser excitation and fluorescence collection efficiency. To relate the recorded image intensity to the number of GFP molecules, the embryos were lysed gently, and a fluorometric analysis of their contents was performed. Confocal laser scanning microscopy data collection from a single sea urchin blastula required less than 2 min, thereby allowing gene expression in dozens of embryos to be monitored in parallel with high spatial and temporal resolution.

Inhibition of Secondary Caries Lesion Progression Using Fluoride Varnish

The purpose of this study was to determine the efficacy of fluoride varnish (FV) in inhibiting progression of secondary caries (SC). In a first experiment, human teeth, restored either with amalgam or resin composite, were exposed for 4 days to a microbial caries model. Half of each specimen was then painted with an acid-resistant nail varnish to maintain the baseline SC lesion. Specimens were then either treated with a FV (removed after 24 h) or not treated (control groups) prior to being exposed for 4 more days to the caries model. A second experiment was conducted to compare the effect that the varnish (with and without fluoride) had on SC development around amalgam. Confocal laser scanning microscopy data from these experiments suggested that the application of a FV to early, active SC lesions significantly slowed down their progression. Furthermore, the application of a placebo varnish showed a trend towards slowing down lesion progression, suggesting that the effect of FV on SC is not only due to its fluoride release.

In situ microscopic analysis of asbestos and synthetic vitreous fibers retained in hamster lungs following inhalation.

Hamsters breathed, nose-only, for 13 weeks, 5 days/week, 6 hr/day, either man-made vitreous fiber (MMVF)10a, MMVF33, or long amosite asbestos at approximately 300 World Health Organization (WHO) fibers/cc or long amosite at 25 WHO fibers/cc. [World Health Organization fibers are longer than 5 microm and thicker than 3 microm, with aspect ratio >3.] After sacrifice, fiber burden was estimated (left lungs) by ashing and scanning electron microscopy (ashing/SEM) or (right middle lobes) by confocal laser scanning microscopy (CLSM) in situ. In situ CLSM also provided three-dimensional views of fibers retained, undisturbed, in lung tissue. Fibers of each type were lodged in alveoli and small airways, especially at airway bifurcations, and were seen fully or partly engulfed by alveolar macrophages. Amosite fibers penetrated into and through alveolar septa. Length densities of fibers in parenchyma (total length of fiber per unit volume of lung) were estimated stereologically from fiber transsections counted on two-dimensional optical sections and were 30.5, 25.3, 20.0, and 81.6 mm/mm3 for MMVF10a, MMVF33, and low- and high-dose amosite, respectively. Lengths of individual fibers were measured in three dimensions by tracking individual fibers through series of optical sections. Length distributions of amosite fibers aerosolized, but before inhalation versus after retention in the lung were similar, whether determined by ashing/SEM or in situ CLSM. In contrast, the fraction of short MMVF10a and MMVF33 fibers increased and the geometric mean fiber lengths of both MMVFs decreased by approximately 60% during retention. Most likely due to fiber deposition pattern and differences in sampling, fiber burdens [MMVF10a, MMVF33, and amosite (high dose; 269 WHO fibers/cc)] determined by ashing/SEM were 1.4, 1. 5, and 3.5 times greater, respectively, than those calculated from in situ CLSM data. In situ CLSM is able to provide detailed information about the anatomic sites of fiber retention and also fiber lengths and burdens in good agreement with ashing/SEM results.

Measurement of Enamel Remineralization Using Microradiography and Confocal Microscopy

Tooth minerals are lost and regained constantly in a normal, human oral environment. Different methods have been developed to measure this gain and loss in enamel minerals; however, these methods deal with different problems, such as being time consuming or involving the use of X–rays. The aim of this study was to determine if remineralization measured in a thin enamel section (TS) by transversal microradiography (MR) can be reliably monitored by measuring lesion parameters (area, total and average dye fluorescence) on the same TS or on half a tooth (HT) with confocal laser scanning microscopy (CLSM). Thirty–six human enamel specimens were demineralized for 96h, and then half of each specimen was covered with an acid–resistant nail varnish. Specimens were divided into three groups (12/group) and subjected for 20 days to a cyclic remineralization regimen with consisted daily of a 4–hour acid challenge, four 1–min treatment periods with 0, 250 or 1,100 ppm F dentifrice slurries (1:2; dentifrice:water) and 20 h in pooled, human saliva, at room temperature. Specimens were cut and analyzed by MR, then stained with a fluorescent dye (0.1mM rhodamine B) for 1 h and analyzed using CLSM. Both MR and CLSM detected significantly greater remineralization (p<0.05) in the specimens treated with the fluoride–containing dentifrices than in the specimens treated with 0 ppm F. Significant differences were detected between specimens treated witht the fluoride–containing dentifrices by MR and CLSM (HT area and total fluorescence). Statistically significant (p<0.05) Pearson correlation coefficients were calculated between the MR and CLSM data: difference in MR mineral content (ΔM) versus HT lesion area = 0.71; ΔM versus HT total fluorescence = 0.70; ΔM versus HT average fluorescence = 0.61; ΔM versus TS lesion area = 0.88; ΔM versus TS total fluorescence = 0.63, and ΔM versus TS average fluorescence = 0.40. It is concluded that confocal microscopy in either TS or HT may provide valid surrogates (area and total fluorescence) for MR measurements in enamel remineralization studies.

Assessment of Cornea Viability by Confocal Laser Scanning Microscopy and MTT Assay

Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness. New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only. This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.

The dynamic nuclear redistribution of an hnRNP K-homologous protein during Drosophila embryo development and heat shock. Flexibility of transcription sites in vivo.

The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.