Combination Of Confocal Laser Scanning Microscopy Research Articles

X-ray computerized microtomography and confocal Raman microscopy as complementary techniques to conventional imaging tools for the microstructural characterization of Cheddar cheese.

This study explored the use of X-ray computerized microtomography (micro-CT) and confocal Raman microscopy to provide complementary information to well-established techniques, such as confocal laser scanning microscopy (CLSM), for the microstructural characterization of cheese. To evaluate the potential of these techniques, 5 commercial Cheddar cheese samples, 3 with different ripening times and 2 with different fat contents, were analyzed. Confocal laser scanning microscopy was particularly useful to describe differences in fat and protein distribution, especially between the 2 samples with different fat contents. The quantitative data obtained through image analysis correlated well with the nutritional information provided in the product labels. Conversely, micro-CT was more advantageous for studying the size and spatial distribution of microcrystals present within the cheese matrix. Two types of microcrystals were identified that differed in size, shape, and X-ray attenuation. The smallest, with a diameter of approximately 10 to 20 μm, were more abundant in the samples and presented a more uniform roundish shape and higher X-ray attenuation. Larger and more heterogeneous crystals with diameters reaching 50 μm were also observed in scarcer numbers and showed lower X-ray attenuation. Confocal Raman microscopy was useful not only for identifying the distribution of all these components but also allowed comparing the presence of micronutrients such as carotenoids in the cheeses and provided compositional information on the crystals detected. Small and large crystals were identified as calcium phosphate and calcium lactate, respectively. Overall, using micro-CT, confocal Raman microscopy, and CLSM in combination generated novel and complementary information for the microstructural and nutritional characterization of Cheddar cheese. These techniques can be used to provide valuable knowledge when studying the effect of milk composition, processing, and maturation on the cheese quality attributes.

Optical and microstructural characterization of Micro-LED with sidewall treatment

The traditional influence on the sidewall damage of Micro-LED was mostly focused on the research of I-V-L. In this paper, we discussed the influence of Micro-LED sidewall damage from the perspective of optical and microstructural characterization. Scanning electron microscopy showed that the Micro-LED structure with smaller size was more irregular after inductively coupled plasma etching. High-resolution transmission electron microscopy and energy dispersive x-ray spectroscopy analysis showed that the area of the upper and lower regions of the quantum well was inconsistent, there was about 2 nm lattice disorder on the surface of the sidewall of the mesa, and oxygen and silicon impurity atoms were enriched. For optical characterization, a method combining laser scanning confocal microscopy and photoluminescence (PL) was proposed to evaluate the optical performance of the mesa. The results showed that the luminescence of Micro-LED mesa was uneven, the luminous intensity at the edge of the mesa was reduced by more than 65%, and the luminous wavelength was shifted by several nanometers. Finally, we optimized the sidewall treatment process, effectively improved the performance of Micro-LED devices by combining tetramethylammonium hydroxide treatment and SiO2 passivation, and increased the luminous intensity of Micro-LED 2 μm away from the edge by about 4.7 times and PL uniformity was greatly improved. These results provided an available reference for the development of Micro-LED.

Relationship Between Antibiotic Resistance, Biofilm Formation, and Biofilm-Specific Resistance in Escherichia coli Isolates from Ningbo, China.

PurposeSeveral Escherichia coli pathotypes still constitute an important public health concern owing to its pathogenicity and antimicrobial resistance. Moreover, biofilm formation of E. coli can allow the strains to interfere with host and antimicrobial eradication, thus conferring additional resistance. The association between the formation of biofilm and antimicrobial resistance determinants has been extensively exploited; nevertheless, there is still no definite conclusion. The purpose of this study was to provide additional data to augment the present knowledge about the subject.MethodsAntibiotic resistance/susceptibility profiles of 81 isolates from pediatric individuals in China between 2011 and 2014 against 20 antibiotics were assessed using the VITEK 2 system. Biofilm-forming capacities were evaluated using the crystal violet staining method, confocal laser scanning microscopy (CLSM), and field emission scanning electron microscopy. Biofilm compositions inside the biofilm formed by representative strains were assessed using CLSM. The effects of antibiotics on biofilms generated by E. coli strains of different biofilm-forming ability were examined using CLSM in combination with gatifloxacin. The relationships between antibiotic resistance, biofilm formation, and biofilm-specific resistance in E. coli isolates were investigated.ResultsThe results showed that 23 isolates were classified as multidrug-resistant, and 57 isolates were classified as extensively drug-resistant (XDR). Among the 69 isolates with the ability to form biofilms, 46 isolates were stronger biofilm formers. Correlation analysis demonstrated that strain populations exhibiting more robust biofilm formation likely contained larger proportions of XDR isolates.ConclusionTogether, our study implies that there was an association between biofilm-formation and resistance to several antibiotics for XDR-E. coli isolates, and would provide novel insights regarding the prevention and treatment against E. coli-related infections.

In vitro visualization and quantitative characterization of Pseudomonas aeruginosa biofilm growth dynamics on polyether ether ketone

Prevention and treatment of orthopedic device-related infection (ODRI) is complicated by the formation of bacterial biofilms. Biofilm formation involves dynamic production of macromolecules that contribute to the structure of the biofilm over time. Limitations to clinically relevant and translational biofilm visualization and measurement hamper advances in this area of research. In this paper, we present a multimodal methodology for improved characterization of Pseudomonas aeruginosa grown on polyether ether ketone (PEEK) as a model for ODRI. PEEK discs were inoculated with P. aeruginosa, incubated for 4-48 htime intervals, and fixed with 10% neutral-buffered formalin. Samples were stained with fluorescent dyes to measure biofilm components, imaged with confocal laser scanning microscopy (CLSM) and scanning electron microscopy(SEM), and quantified.We were able to visualize and quantify P. aeruginosa biofilm growth on PEEK implants over 48 h. Based on imaging data, we propose a generalized growth cycle that can inform orthopedic diagnostic and treatment for this pathogen on PEEK. These results demonstrate the potential of using a combined CLSM and SEM approach for determining biofilm structure, composition, post-adherence development on orthopedic materials. This model may be used for quantitative biofilm analysis for other pathogens and other materials of orthopedic relevance for translational study of ODRI.

A novel non-destructive technique for qualitative and quantitative measurement of dental erosion in its entirety by porosity and bulk tissue-loss

ObjectiveTo explore the potential of combining non-contact profilometry (NCP) and confocal laser scanning microscopy (CLSM) data to measure the entire erosive process non-destructively and to validate findings using inductively coupled plasma-atomic emission spectroscopy (ICP-AES), scanning electron microscopy (SEM) and surface microhardness (SMH) using the same samples throughout. MethodsPolished bovine enamel samples (n = 35) were divided into groups (7/group) with similar SMH values. Samples underwent individual erosive challenges (1 % citric acid, pH3.8) for 1, 5, 10, 15 or 30 min under stirring and aliquot extracts were analysed for Ca and P by ICP-AES. SMH was used to measure erosive softening. Profilometry was used to assess bulk volume loss (BVL). Images were captured by SEM. Samples were stained with rhodamine-B (0.1 mM, 24 h) and images captured by CLSM. Image processing was used to determine changes in fluorescent volume for the first 10 μm (ΔFV10) for each enamel sample which were combined with BVL to calculate total lesion volume (TLV). ANOVA, linear regression and Pearson correlation analysis were used where applicable. ResultsSurface softening, [Ca], [P], BVL and ΔFV10μm increased with acid erosion duration which were significant by 10 min (P < .01). The Ca:P ratio increased to 1.57 then decreased after 5 min erosion suggesting a sub/surface phase change, which was observed by SEM and CLSM showing significant changes to the enamel surface and subsurface morphology with time. Combination of BVL and ΔFV10 as TLV strengthened the significant correlations with [Ca], [P], and SMH (P < .01). ConclusionThis novel combination of CLSM and NCP allows for concurrent non-destructive quantification of the entire erosive process by mineral loss, and qualitatively characterise microstructural changes during dental erosion.

Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.

Monoterpenes-containing PEGylated transfersomes for enhancing joint cavity drug delivery evidenced by CLSM and double-sited microdialysis.

The synovial tissues are natural sites of drug delivery for the treatment of rheumatoid arthritis. Our previous study showed that mixed monoterpenes edge-activated PEGylated transfersomes (MMPTs) could significantly enhance the percutaneous absorption of sinomenine (SIN), an anti-inflammation drug. The aim of this study was to investigate the potential of MMPTs for delivery of SIN to the synovial tissues in joint cavities. To this end, conventional liposomes (LPSs) were used as a reference. Transmission electron microscope, constant pressure extrusion method, and differential scanning calorimetry (DSC) were used for physicochemical characterization of the formulations. Confocal laser scanning microscopy (CLSM) and double-sited microdialysis coupled with LC-MS/MS were exploited to study the distribution of MMPTs in different skin layers and pharmacokinetics of SIN in the blood and the joint cavities. The results showed that mixed monoterpenes could significantly enhance the elasticity of MMPTs, evidenced by a decrease in the main transition temperature (Tm) and transition enthalpy (△H). CLSM analyses demonstrated that MMPTs were distributed in deep layers of the skin, indicating that MMPTs might transport SIN through the skin. In contrast, LPSs were confined in the stratum corneum, which deterred SIN from penetrating through the skin. The results from double-sited microdialysis pharmacokinetics showed that in the joint cavities the steady state concentration (Css) and AUC0→t of SIN from MMPTs were 2.1-fold and 2.5-fold of those from LPSs, respectively. In contrast, in the blood the Css and AUC0→t of SIN from MMPTs were about 1/3 of those from LPSs. This study suggested that MMPTs could enhance the delivery of SIN to the joint cavities. A combination of CLSM and double-sited microdialysis could give an insight into the mechanism of transdermal and local drug delivery.

Direct Observation of Gas Meniscus Formation on a Superhydrophobic Surface.

The formation of a bridging gas meniscus via cavitation or nanobubbles is considered the most likely origin of the submicrometer long-range attractive forces measured between hydrophobic surfaces in aqueous solution. However, the dynamics of the formation and evolution of the gas meniscus is still under debate, in particular, in the presence of a thin air layer on a superhydrophobic surface. On superhydrophobic surfaces the range can even exceed 10 μm. Here, we report microscopic images of the formation and growth of a gas meniscus during force measurements between a superhydrophobic surface and a hydrophobic microsphere immersed in water. This is achieved by combining laser scanning confocal microscopy and colloidal probe atomic force microscopy. The configuration allows determination of the volume and shape of the meniscus, together with direct calculation of the Young-Laplace capillary pressure. The long-range attractive interactions acting on separation are due to meniscus formation and volume growth as air is transported from the surface layer.

Colloidal liquid crystals in square confinement: isotropic, nematic and smectic phases

We report on the confinement of colloidal liquid crystals in three dimensional chambers with a square footprint. To this end we use colloidal silica rods and exploit their relatively large density difference with respect to the dispersing solvent to study isotropic, nematic and smectic phases confined into a single chamber. Combining laser scanning confocal microscopy and soft-lithography techniques enables us to characterize the configurations down to the single particle level. We will focus on the smectic phase and compare to recent theories and simulations.

Effects of roasting temperatures and grinding type on the yields of oil and protein obtained by aqueous extraction processing

A three cylindrical roll crusher was used in this study to solve the comminution problem and to improve oil and protein yields in aqueous extraction processing (AEP) of peanuts. We combined confocal laser scanning microscopy (CLSM) and particle size distribution analysis to investigate the effect of different peanut processing material on oil and protein extraction. A proper roasting treatment (150 °C) is beneficial to oil extraction yield. However, the protein yield has been declining from 84.33% to 51.40% with the increase of roasting temperature from (130 °C–210 °C). The optimal average particle size of peanut paste in AEP was 15.2 μm which could hardly find intact oil bodies by CLMS. Nevertheless, the remained intact of protein body still could be found in insoluble fraction. In AEP, highest free oil yield (92.2%) was achieved with roasted peanut (150 °C, 20 min) using 1: 5 solid to liquid ratio (twice ground peanut pastes/water), pH 9, 60 °C for 2 h and demusification by adding 0.5% (w/w) Protex 50FP at pH 4.5 and incubating at 50 °C for 2 h. Without roasting treatment, the oil contents of insoluble and residual cream fractions from AEP were highly correlated with their protein contents (R2 = 0.8724, 0.9178, respectively). Additionally, the exposure of interior hydrophobic groups of peanut protein adhere more oil to the protein body surface, which may have led to increment in the oil content of insoluble fraction.

3D imaging of cement-based materials at submicron resolution by combining laser scanning confocal microscopy with serial sectioning.

In this paper, we present a new method to reconstruct large volumes of nontransparent porous materials at submicron resolution. The proposed method combines fluorescence laser scanning confocal microscopy with serial sectioning to produce a series of overlapping confocal z-stacks, which are then aligned and stitched based on phase correlation. The method can be extended in the XY plane to further increase the overall image volume. Resolution of the reconstructed image volume does not degrade with increase in sample size. We have used the method to image cementitious materials, hardened cement paste and concrete and the results obtained show that the method is reliable. Possible applications of the method such as three-dimensional characterization of the pores and microcracks in hardened concrete, three-dimensional particle shape characterization of cementitious materials and three-dimensional characterization of other porous materials such as rocks and bioceramics are discussed.

Effect of polyethyleneglycol (PEG) chain length on the bio–nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells

When nanoparticles (NPs) enter a physiological environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) analysis allowed us to accurately determine the corona composition showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 in order to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry analysis in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.

New Bone Formation and Microstructure Assessed by Combination of Confocal Laser Scanning Microscopy and Differential Interference Contrast Microscopy

Bone is a mineralized connective tissue that is continuously and microstructurally remodeled. Altered bone formation and microstructure arise in pathological bone conditions such as osteoporosis, osteonecrosis, fracture repair, and Paget disease of bone. A proper and objective assessment of bone formation and microstructure will provide insight into the understanding of bone pathogenesis and remodeling. Here, new bone formation ex vitro and its microstructure were evaluated in in vivo multiple sequential polychrome-labeled samples using confocal laser scanning microscopy (CLSM), which generated clearer and more reliable images of thick bone sections than conventional fluorescence microscopy (CFM). Intriguingly, fine details of the bone microstructural features, including the mineralization fronts, quiescent versus active osteons, and Volkmann's channel, were elucidated using CLSM, which defines the relationship between morphological changes and function, when combined with differential interference contrast microscopy. Furthermore, CLSM provided objective evaluations of bone formation, such as the ratio of labeled areas of new bone formation in a rabbit model when compared with CFM. Altogether, new bone formation and its microstructure can be evaluated more adequately using a combination of CLSM and DIC microscopies.

Correlation Between the Microstructural Development of Bainitic Ferrite and the Characteristics of Martensite-Austenite Constituent

Microstructural development of bainitic ferrite under a simulated weld heat-affected zone (HAZ) of low-carbon steel was correlated with the characteristics of martensite-austenite (M-A) constituent. By combining laser scanning confocal microscopy (LSCM) observations and electron backscatter diffraction (EBSD) analysis, we evaluated variant pair formation in time-series, which was related to the second-phase formation phenomenon. Furthermore, the classification of the microstructural development showed that the characteristics of M-A in a weld HAZ can be predicted from the microstructural development of bainitic ferrite.

Time Evolution of Nanoparticle–Protein Corona in Human Plasma: Relevance for Targeted Drug Delivery

When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as "protein corona". This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle's lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol-DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol-DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C-II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol-DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

Volume 81A, Number 2 February 2012 Cover Image

AbstractOn the cover: Zebrafish vasculature portrayed in a whole new light on the cover of this issue. The source work by Zhang and coworkers in the current issue describes an integrated optical system, combining laser scanning confocal microscopy and in vivo flow cytometry, to track cells (both stationary and circulating) in zebrafish.Cover design by Bärbel Beran [www.beran‐design.de].

Biofilm formation in environmental bacteria is influenced by different macromolecules depending on genus and species

The formation of biofilms by diverse bacteria isolated from contaminated soil and groundwater on model substrata with different surface properties was assessed in a multifactorial screen. Diverse attachment phenotypes were observed as measured by crystal violet dye retention and confocal laser scanning microscopy (CLSM). Bulk measurements of cell hydrophobicity had little predictive ability in determining whether biofilms would develop on hydrophobic or hydrophilic substrata. Therefore selected pairs of bacteria from the genera Rhodococcus, Pseudomonas and Sphingomonas that exhibited different attachment phenotypes were examined in more detail using CLSM and the lipophilic dye, Nile Red. The association of Rhodococcus sp. cell membranes with lipids was shown to influence the attachment properties of these cells, but this approach was not informative for Pseudomonas and Sphingomonas sp. Confocal Raman Microspectroscopy of Rhodococcus biofilms confirmed the importance of lipids in their formation and indicated that in Pseudomonas and Sphingomonas biofilms, nucleic acids and proteins, respectively, were important in identifying the differences in attachment phenotypes of the selected strains. Treatment of biofilms with DNase I confirmed a determining role for nucleic acids as predicted for Pseudomonas. This work demonstrates that the attachment phenotypes of microbes from environmental samples to different substrata varies markedly, a diverse range of macromolecules may be involved and that these differ significantly between genera. A combination of CLSM and Raman spectroscopy distinguished between phenotypes and could be used to identify the key macromolecules involved in cell attachment to surfaces for the specific cases studied.

Combined application of 13C NMR spectroscopy and confocal laser scanning microscopy—Investigation on biofilm structure and physico-chemical properties

Long-term biofilm processes are influenced by the interplay of biofilm accumulation and detachment, which in turn depend partially on the biofilm structure and composition. In this study a combination of confocal laser scanning microscopy (CLSM) and nuclear magnetic resonance (NMR) spectroscopy was applied to analyze biofilm structure, composition and molecular mobility. Whereas CLSM delivers information about the structure of biofilms the NMR measurement provides detailed but not locally resolved information about the chemical composition of biofilm constituents. Heterotrophic mixed-species biofilms were cultivated in rotating annular reactors exposed to different flow conditions and glucose concentrations in order to obtain biofilms with diverse architectural structures. The growth state of the biofilms appeared to influence the composition of biofilm and detached biomass. The difference in the 13C NMR spectra between the differently structured biofilms or between biofilm and detached biomass was small, except for the still exponential growing biofilm supplied with the highest glucose concentration. More information was gained from the mobility of specific molecular groups within the biofilm biomass. Molecules within the biofilm biomass of the non-filamentous biofilms were more strongly bound than the molecules within the respective detached biomass. Glucose starvation resulted in a reduction in the biofilm molecular mobility. The opposite was observed in the filamentous biofilm. In this case, the molecular mobility in the biofilm increased after starvation and the molecules in the detached biomass were bound more strongly than in the respective biofilm biomass. It could be shown that the combination of CLSM and 13C NMR spectroscopy is a promising approach to analyze the interactions between biofilm architecture, composition or growth state and biofilm detachment.

Quantifying cellular traction forces in three dimensions

Cells engage in mechanical force exchange with their extracellular environment through tension generated by the cytoskeleton. A method combining laser scanning confocal microscopy (LSCM) and digital volume correlation (DVC) enables tracking and quantification of cell-mediated deformation of the extracellular matrix in all three spatial dimensions. Time-lapse confocal imaging of migrating 3T3 fibroblasts on fibronectin (FN)-modified polyacrylamide gels of varying thickness reveals significant in-plane (x, y) and normal (z) displacements, and illustrates the extent to which cells, even in nominally two-dimensional (2-D) environments, explore their surroundings in all three dimensions. The magnitudes of the measured displacements are independent of the elastic moduli of the gels. Analysis of the normal displacement profiles suggests that normal forces play important roles even in 2-D cell migration.

Morphological and crystallographic evolution of bainite transformation in Fe‐0.15C binary alloy

In this article, an in situ observation method, combining laser scanning confocal microscopy and electron backscattering diffraction, was used to investigate the morphological and crystallographic evolution of bainite transformation in a Fe-0.15C binary alloy. The nucleation at a grain boundary and inclusions, sympathetic nucleation, and impingement event of bainitic ferrite were directly shown in real time. The variant evolution during bainite transformation and misorientation between bainitic ferrites were clarified. Strong variant selection was observed during sympathetic nucleation.