Analysis of fluorescence from algae fossils of the Neoproterozoic Doushantuo formation of China by confocal laser scanning microscope

Chinese algae fossils can provide unique information about the evolution of the early life. Thin sections of Neoproterozoic algae fossils, from Guizhou, China, were studied by confocal laser scanning microscopy, and algae fossils were fluorescenced at different wavelengths when excited by laser light of 488 nm, 476 nm, and 568 nm wavelength. When illuminated by 488 nm laser light, images of the algae fossils were sharper and better defined than when illuminated by 476 nm and 568 nm laser light. The algae fossils fluoresce at a wide range of emission wavelengths. The three-dimensional images of the fluorescent algae fossils were compared with the transmission images taken by light microscope. We found that the fluorescence image of the confocal laser scanning microscope in a single optical section could pass for the transmission image taken by a light microscope. We collected images at different sample depths and made a three-dimensional reconstruction of the algae fossils. And on the basis of the reconstruction of the three-dimensional fluorescent images, we conclude that the two algae fossils in our present study are red algae.     

Evaluation of epoxy resin sealer after three root canal filling techniques by confocal laser scanning microscopy

The aim of this study was to evaluate the penetration of endodontic sealer into the dentin tubules, the integrity of the sealer layer perimeter, and the sealer area at the apical third after different filling techniques by confocal laser scanning microscopy (CLSM). Forty‐five mandibular premolars were mechanically prepared with ProTaper files, until F5 file. Thereafter, they were filled with an epoxy‐resin sealer (AH Plus) mixed with Rhodamine B dye (0.1% proportion) and allocated in three groups: Group 1, single master cone; Group 2, cold lateral compaction; and Group 3, Thermafil. For confocal laser scanning microscopy analysis, the specimens were transversely sectioned at 4 mm from the apex. The images at ×10 and ×40 were analyzed by Imagetool 3.0 software. Significant differences were not found among the three experimental groups according the dentin‐impregnate area by the sealer (P = 0.68) and between the sealer and root canal perimeter (P = 0.18). However, root canal filling techniques were significantly different when apical sealer areas were compared (P = 0.001). Thermafil group showed smaller sealer areas (8.09%) while cold lateral compaction and gutta‐percha master cone showed similar areas (17.37 and 21.18%, respectively). The dentin‐impregnated area was not dependent on the root canal filling technique. Single master cone, cold lateral condensation and Thermafil techniques presented integrity of the sealer perimeter close to 100% and Thermafil resulted in a significantly thinner sealer layer. Microsc. Res. Tech. 75:1277–1280, 2012. © 2012 Wiley Periodicals, Inc.     

Three‐dimensional imaging of porous media using confocal laser scanning microscopy

In the last decade, imaging techniques capable of reconstructing three‐dimensional (3‐D) pore‐scale model have played a pivotal role in the study of fluid flow through complex porous media. In this study, we present advances in the application of confocal laser scanning microscopy (CLSM) to image, reconstruct and characterize complex porous geological materials with hydrocarbon reservoir and CO₂ storage potential. CLSM has a unique capability of producing 3‐D thin optical sections of a material, with a wide field of view and submicron resolution in the lateral and axial planes. However, CLSM is limited in the depth (z‐dimension) that can be imaged in porous materials. In this study, we introduce a ‘grind and slice’ technique to overcome this limitation. We discuss the practical and technical aspects of the confocal imaging technique with application to complex rock samples including Mt. Gambier and Ketton carbonates. We then describe the complete workflow of image processing to filtering and segmenting the raw 3‐D confocal volumetric data into pores and grains. Finally, we use the resulting 3‐D pore‐scale binarized confocal data obtained to quantitatively determine petrophysical pore‐scale properties such as total porosity, macro‐ and microporosity and single‐phase permeability using lattice Boltzmann (LB) simulations, validated by experiments.     

Multipoint scanning dual‐detection confocal microscopy for fast 3D volumetric measurement

We propose a multipoint scanning dual‐detection confocal microscopy (MS‐DDCM) system for fast 3D volumetric measurements. Unlike conventional confocal microscopy, MS‐DDCM can accomplish surface profiling without axial scanning. Also, to rapidly obtain 2D images, the MS‐DDCM employs a multipoint scanning technique, with a digital micromirror device used to produce arrays of effective pinholes, which are then scanned. The MS‐DDCM is composed of two CCDs: one collects the conjugate images and the other collects nonconjugate images. The ratio of the axial response curves, measured by the two detectors, provides a linear relationship between the height of the sample surface and the ratio of the intensity signals. Furthermore, the difference between the two images results in enhanced contrast. The normalising effect of the MS‐DDCM provides accurate sample heights, even when the reflectance distribution of the surface varies. Experimental results confirmed that the MS‐DDCM achieved high‐speed surface profiling with improved image contrast capability.     

Three-dimensional visualization and physiologic evaluation of bile canaliculi in the rat liver slice by confocal laser scanning microscopy.

We evaluated the morphology and physiologic function of the bile canaliculi (BC) in the rat liver slice (RLS) by confocal laser scanning microscopy (CLSM). Lucifer yellow (LY) dye was injected into the RLS, and the distribution of LY was serially evaluated. After the injection of LY, hepatocytes were initially visualized, followed by visualization of the BC. There was no significant difference in the distribution of LY between zones 1 and 3 in the hepatic lobule. In zone 1, the reticular distribution of the BC was observed, whereas the part of BC was linearly visualized in zone 3 along the course of sinusoids. When changes in the bile canalicular fluorescence (BCF) were serially evaluated, the BCF was decreased to the minimal level (88% of the value obtained immediately after the LY injection) 10 min after the LY injection, and it tended to increase thereafter. The intralobular hepatocyte fluorescence (ILHF) was decreased to 58.9% of the initial value during the first 40 min. However, the ILHF was transiently increased 30 min after the LY injection, suggesting the possibility of reabsorption of LY by hepatocytes. Three-dimensional (3-D) reconstruction images of the BC facilitated the evaluation of the stereoscopic structure of BC. Confocal laser scanning microscopy facilitated the evaluation of structures and physiologic function of the BC.     

Real‐time in vivo confocal laser scanning microscopy of melanin‐containing cells: A promising diagnostic intervention

The use of noninvasive imaging techniques to evaluate different types of skin lesions is increasing popular. In vivo confocal laser scanning microscopy (CLSM) is a new method for high resolution non‐invasive imaging of intact skin in situ and in vivo. Although many studies have investigated melanin‐containing cells in lesions by in vivo CLSM, few studies have systematically characterized melanin‐containing cells based on their morphology, size, arrangement, density, borders, and brightness. In this study, the characteristics of melanin‐containing cells were further investigated by in vivo CLSM. A total of 130 lesions, including common nevi, giant congenital pigmented nevi, vitiligo, melasma, melanoma, and chronic eczema, were imaged by in vivo CLSM. This research helps dermatologists understand the characteristics of melanin‐containing cells and facilitate the clinical application of melanin‐containing cells in the investigation of dermatological disease. In summary, melanin‐containing cells include keratinocytes, melanocytes, macrophages, and melanocytic skin tumor cells. Our study presents the CLSM characteristics of melanin‐containing cells to potentially facilitate in vivo diagnosis based on shape, size, arrangement, density, borders, and brightness. Microsc. Res. Tech. 78:1121–1127, 2015. © 2015 Wiley Periodicals, Inc.     

Three‐dimensional visualization of dermal skin structure using confocal laser scanning microscopy

The properties and performance of collagen‐based materials may be affected by the collagen fibre bundle pattern, orientation and weave. The aim of this study was to develop and apply methods to visualize the dermis using confocal laser scanning microscopy from thin tissue sections stained with haematoxylin and eosin. The data was processed to allow three‐dimensional (3‐D) visualization on a PC and using a 3‐D immersive technology system. The 3‐D visualization of the confocal microscope image stacks allowed the evaluation of the collagen macromolecular structure including the collagen fibre bundles. The methods developed provide a novel way of viewing complex organic structures with further potential applications in the medical field.     

Quantitative tomography of early mouse embryos: laser scanning microscopy and 3D reconstruction

The cell volume alteration participates in a wide variety of cellular functions that may interfere with intra‐cellular homeostasis. The most adequate approach of estimation of the volume changes induced by osmotic misbalance, alteration in shape and size due to the action shape forming substances, etc., is the direct measurement of volumetric parameters of embryos. In the given research, the volume magnitude and kinetics of changes in volume and surface area of blastomere and polar bodies of early mouse embryos were determined using three‐dimensional reconstruction of the optical section stack obtained with laser scanning microscope (LSM). The size and surface area were determined for isotonic and anisosmotic conditions. The physiological significance of the findings is discussed.     

Three‐dimensional morphology of cerebellar climbing fibers. A study by means of confocal laser scanning microscopy and scanning electron microscopy

The intracortical pathway of cerebellar climbing fibers have been traced by means of scanning electron microscpy (SEM) and confocal laser scanning microscopy (CLSM) to study the degree of lateral collateralization of these fibers in the granular Purkinje cell and molecular layers. Samples of teleost fish were processed for conventional and freeze‐fracture SEM. Samples of hamster cerebellum were examined by means of CLSM using FM4–64 as an intracellular stain. High resolution in lens SEM of primate cerebellar cortex was carried out using chromium coating. At scanning electron and confocal laser microscopy levels, the climbing fibers appeared at the white matter and granular layer as fine fibers with a typical arborescence or crossing‐over branching pattern, whereas the mossy fibers exhibited a characteristic dichotomous bifurcation. At the granular layer, the parent climbing fibers and their tendrils collaterals appeared to be surrounding granule and Golgi cells. At the interface between granule and Purkinje cell layers, the climbing fibers were observed giving off three types of collateral processes: those remaining in the granular layer, others approaching the Purkinje cell bodies, and a third type ascending directly to the molecular layer. At this layer, retrograde collaterals were seen descending to the granular layer. By field emission high‐resolution SEM of primate cerebellar cortex, the climbing fiber terminal collaterals were appreciated ending by means of round synaptic knobs upon the spines of secondary and tertiary Purkinje cell dendrites.     

Penetrability of AH plus and MTA fillapex after endodontic treatment and retreatment: A confocal laser scanning microscopy study

The aim of the study was to assess the penetrability of two endodontic sealers (AH Plus and MTA Fillapex) into dentinal tubules, submitted to endodontic treatment and subsequently to endodontic retreatment. Thirty ex vivo incisors were prepared using ProTaper rotary system up to F3 instrument and divided in three groups according to the endodontic sealer used for root canal filling: AH Plus (AHP), MTA Fillapex (MTAF), and control group (CG) without using EDTA previously to the root canal filling. Rhodamine B dye (red) was incorporated to the sealers in order to provide the fluorescence which will enable confocal laser scanning microscopy (CLSM) assessment. All specimens were filled with gutta‐percha cones using the lateral compaction technique. The specimens were submitted to endodontic retreatment using ProTaper Retreatment system, re‐prepared up to F5 instruments and filled with gutta‐percha cones and the same sealer used during endodontic retreatment. Fluorescein dye (green) was incorporated to the sealer in order to distinguish from the first filling. The roots were sectioned 2 mm from the apex and assessed by CLSM. No difference was found between the two experimental groups (P > 0.05). On the other hand, in the control group the sealers were not capable to penetrate into dentinal tubules after endodontic treatment (P > 0.05). In retreatment cases, none of the sealers were able to penetrate into dentin tubules. It can be concluded that sealer penetrability is high during endodontic treatment. However, MTA Fillapex and AH Plus do not penetrate into dentinal tubules after endodontic retreatment. Microsc. Res. Tech. 77:467–471, 2014. © 2014 Wiley Periodicals, Inc.     

Dentinal tubule penetration of endodontic sealers after nonthermal plasma treatment: A confocal laser scanning microscopy study

One of the factors affecting the success of endodontic treatment is to fill the root canal system hermetically. The aim of this in vitro study was to evaluate the effect of nonthermal plasma (NP) on dentinal tubule penetration of root canal sealers using confocal laser scanning microscopy. Forty mandibular premolar teeth were selected and the root canals were prepared with large‐Waveone‐Gold rotary‐files. Specimens were divided into four experimental groups according to sealer and NP treatment (n = 10). G1: AH‐Plus (AH) G2: nonthermal plasma application + AH‐Plus(AH‐P) G3: Endosequence‐BC(BC) G4: nonthermal plasma application + Endosequence‐BC(BC‐P). Cold lateral‐condensation technique was used for the obturation of root canals. The roots were sectioned horizontally and the sections were examined under confocal laser scanning microscopy. The maximum tubule penetration and percentage of penetration values were obtained from the microscopy images and were statistically analyzed with repeated measurements‐ANOVA and the Tukey (HSD) test (p < 0.05). The percentages of dentinal tubule penetration of the groups were not statistically different. The maximum tubule penetration of the AH‐P was statistically lower than that of the BC‐P (p < 0.05). Plasma application had no affect on the percentage of dentinal tubule penetration. Under the conditions of this in vitro Endosequence‐BC sealer showed higher maximum tubule penetration values than AH‐Plus after NP treatment. Percentage of dentinal tubule penetration values of experimental groups was similar.     

The effect of different obturation methods on sealer penetration alongside apically separated rotary nickel–titanium instruments: A confocal laser scanning microscopy study

The effects of different obturation techniques on calcium silicate‐based sealer penetration in the presence of apically separated rotary files were evaluated. Forty‐eight extracted mandibular incisors were used. ProTaper F2 rotary files were separated at the apical thirds. Samples were divided into four groups (n = 12) according to obturation technique used: (a) cold lateral compaction (CLC); (b) single cone; (c) bulk‐fill (BF) without a core material; and (d) thermoplastic injection (TI). Specimens were sectioned horizontally at 1 and 3 mm from the apex and studied using a confocal scanning laser microscope. The maximum tubule penetration depth and percentage of penetration were measured. Data were statistically analyzed using parametric and nonparametric tests with a significance level of 5%. Regarding penetration depth, a significant difference was found at 1 mm (p < .05), while no significant difference was found at 3 mm (p > .05). At the 3 mm level, all of the obturation techniques showed similar penetration depths. Regarding penetration percentage, the values of the CLC and TI groups were statistically less when compared with the BF group at 1 and 3 mm levels, respectively (p < .05). Under the limitations of this in vitro study, results suggest that the obturation technique may present a significant effect on sealer penetration.     

New data‐driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume

Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex‐shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex‐shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data‐driven formula‐based approach to the calculation of biovolume of this complex‐shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.     

Dimensional metrology of lab‐on‐a‐chip internal structures: a comparison of optical coherence tomography with confocal fluorescence microscopy

The characterization of internal structures in a polymeric microfluidic device, especially of a final product, will require a different set of optical metrology tools than those traditionally used for microelectronic devices. We demonstrate that optical coherence tomography (OCT) imaging is a promising technique to characterize the internal structures of poly(methyl methacrylate) devices where the subsurface structures often cannot be imaged by conventional wide field optical microscopy. The structural details of channels in the devices were imaged with OCT and analyzed with an in‐house written ImageJ macro in an effort to identify the structural details of the channel. The dimensional values obtained with OCT were compared with laser‐scanning confocal microscopy images of channels filled with a fluorophore solution. Attempts were also made using confocal reflectance and interferometry microscopy to measure the channel dimensions, but artefacts present in the images precluded quantitative analysis. OCT provided the most accurate estimates for the channel height based on an analysis of optical micrographs obtained after destructively slicing the channel with a microtome. OCT may be a promising technique for the future of three‐dimensional metrology of critical internal structures in lab‐on‐a‐chip devices because scans can be performed rapidly and noninvasively prior to their use.     

Application of confocal scanning laser microscopy for an automated nuclear grading of prostate lesions in three dimensions

Confocal scanning laser microscopy provides the opportunity to obtain three-dimensional (3-D) images by piling up consecutive confocal planes. This technique was applied to capture 3-D images from 100-μm-thick tissue blocks from prostate lesions (hyperplasia, dysplasia, adenocarcinomas). Automated methods were implemented to perform a nuclear grading of 3-D cell nuclei from these specimens. Special attention was focused on the development of a new approach to 3-D chromatin texture analysis. This method uses mathematical morphology operations to tessellate the chromatin into homogeneous domains. The nuclear features (volume, shape, texture) were subjected to a discriminant analysis. Using a set of five features, the classification of cell nuclei yielded an accuracy of 963%. The results indicate the potential of 3-D imaging and analysis techniques for an automated nuclear grading of prostate lesions.     

Morphological and confocal laser scanning microscopic investigations of the adductor muscle‐shell interface in scallop

The challenge of joining dissimilar advanced materials has led researchers around the world to search for new and more efficient solutions. This way, we can highlight the muscle‐shell attachment in mollusk, which possessed high strength and toughness. In order to make clear how this “bi‐material interface” derives its superior mechanical properties, the morphological features of the adductor muscle scar in Patinopecten yessoensis was investigated by means of confocal laser scanning microscopy (CLSM). This scar area was found to consist of a myostracum with many evenly distributed pit structures and a fracture section with a parallel arranged prism‐like structure. The measured values of the distribution density, diameter, and depth of those pit structures were 24 , 7.36 2.47 , and 1 0.31 respectively. Profile of each pit wall was arc curve without closed angle. Furthermore, CLSM micrographs showed that considerable micro pits (0.1–0.9 in diameter) distribute round the pit wall and on the pit bottom. This special micromorphology is the first report on the adductor muscle scar in scallop. In addition, the mineral state and mechanical property of the scar surface was analyzed by XRD and nanoindentation test respectively. In general, the study results presented in this work elucidated that the adductor muscle of P. yessoensis was attached to the shell by insertion of collagen fibers and fibril bundles branched from themselves into pits on the myostracum. This specific connection mechanism can increase the strength of the interface without compromising its ductility and toughness. Microsc. Res. Tech. 78:761–770, 2015. © 2015 Wiley Periodicals, Inc.     

Registration of confocal scanning laser microscopy and quantitative backscattered electron images for the temporospatial quantification of mineralization density in 18-month old thoroughbred racehorse articular calcified cartilage

Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (Dm) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine Dm, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and Dm data. The repeatability of the registration and measurement protocols was determined. Dm profiles between calcein labels were explored for an association with time. Dm was 119.7 +/- 24.5 (mean +/- standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 microm/day, respectively. Coefficients of variation (CV) for Dm were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between Dm and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.     

Confocal laser scanning microscopy – an attractive tool for studying the uptake of xenobiotics into plant foliage

The application of confocal laser scanning microscopy (CLSM) to the study of xenobiotic uptake into plant foliage is explored in this paper. Three fluorescent dyes of low molecular weight and contrasting polarities (hydrophilic, moderately lipophilic and lipophilic) were selected to represent foliage‐applied pesticides. These model compounds were applied as droplets to the surfaces of various leaves and/or fruits according to the particular experiment. The transcuticular diffusion behaviour, the compartmentation into epidermal cells and the influence of a surfactant on the uptake of these fluorescent compounds were visualized by CLSM. Distinct differences in diffusion speed across the cuticle and distribution in cell compartments were found between different fluorescent compounds. The presence of a surfactant significantly accelerated the uptake of the moderately lipophilic dye into both thin‐ and thick‐cuticled leaves. The results are discussed in relation to the current knowledge on pesticide uptake and translocation. The advantages and limitations of this technique are highlighted.