Light Fluorescence Microscopy Research Articles

Comparative study of remineralization potential of three different remineralizing agents on demineralized enamel using light fluorescence and confocal fluorescence microscope: An in vitro study

Aim and Objective: The main objective of this in vitro study was to evaluate the comparative analysis of remineralization potential of three different materials – casein phosphopeptide-amorphous calcium phosphate with fluoride (CPP-ACPF), calcium sucrose phosphate (CaSP), and bioactive glass on demineralized enamel using light fluorescence microscopy and confocal laser scanning fluorescence microscopy. Materials and Methods: A total of 40 single-rooted maxillary and mandibular premolars were selected; 4 mm × 4 mm window was prepared on the buccal surfaces of the teeth, which was then subjected to demineralization for 96 h at 37°C. Teeth were randomly selected and divided into four study groups of 10 teeth each: Group 1 (artificial saliva), Group 2 (CPP-ACPF), Group 3 (bioactive glass), and Group 4 (CaSP). Each group was treated with respective remineralizing agents and sectioned with Struers Minitom diamond saw. Each section obtained was visualized under light fluorescence microscope for detection of remineralized and demineralized zones and also was visualized under confocal laser scanning fluorescent microscope for the quantification of demineralized and remineralized zones. Statistical Analysis: Statistical analysis was done using paired t-test, followed by one-way ANOVA, where P ≤ 0.05. Results: All the groups showed better statistically significant remineralization potential when compared to the control group, but among them Group 4, that is, Toothmin group showed the highest mean remineralized value, followed by Groups 3, 2, and 1, though the values were not statistically significant (P ≤ 0.05). Light fluorescence microscopy was an efficient diagnostic aid in detecting remineralization and demineralization. Conclusion: CaSP (Toothmin) has got the best remineralization potential when compared to other groups.

Protoperidinium hormusum sp. nov. (Dinoflagellata) from the Strait of Hormuz (Iran)

A new species of dinoflagellate, Protoperidinium hormusum sp. nov. (Dinoflagellata, Protoperidiniaceae), was described by means of light fluorescent microscopy. It was found in the plankton of the Strait of Hormuz, connecting the Oman and Persian Gulfs, in 2015. Cells of P. hormusum have a five-pointed star form, are symmetrical, and moderately flattened dorsoventrally; antapical horns are widely separated, relatively thin, and divergent. The epicone is conical with an apical pore. The first apical plate 1' is ortho and the second anterior intercalary plate 2a is neutra or hexa. The cingulum is median, slightly ascending (by 0.1-0.2 μm of its width). The sulcus is slightly extending onto the epicone, with an elongated oval indentation, not reaching the antapex. Cell dimensions: length is 86.7-104.4 μm (95.7 ± 5.18 μm) and width is 64.9-81.6 μm (72.0 ± 5.22 μm). New species resembles P. divaricatum and P. conicum in the cells' general outline and dimensions, and the type of the first apical and second intercalary plates but differs from them in a more pronounced pentagonal shape, greater symmetry of cells, and appearance of antapical horns.

Interaction of wheat macromolecules and berry pomace in model dough: Rheology and microstructure

The incorporation of dried black currant pomace (BCP) in wheat dough is a possible way to increase the sustainability of the production chain by adding value to a by-product from juice processing that is up to now regarded as waste. The partial replacement of wheat flour by dried black currant pomace (BCP) increased the water absorption of dough and dough development time. Small strain oscillatory rheology showed that dough stiffness increased with increasing levels of pomace addition (G' at 1 Hz was 8.2 kPa for the reference, and 49.7 kPa for dough with 30% BCP). This result is supported by the higher dough stability observed in farinograms. After simulated baking, dough with 30% BCP showed higher storage and loss moduli compared to the reference and to dough with lower levels of BCP. The increased structural strength that comes with increasing pomace levels is explained by interactions with gluten matrix through hydrogen bonds. The gluten is less developed through the existence of BCP which results in a lower stickiness, resistance and extensibility of the dough. Scanning electron microscopy and fluorescence light microscopy images showed the disruption of gluten film by fibre incorporation. Considering those influences on dough properties, it can be reasoned that dough with 10% BCP would produce products with satisfying characteristics, whereas higher application levels cannot be recommended without using additives to increase the gluten strength.

The Role of Nephronectin on Proliferation and Differentiation in Human Dental Pulp Stem Cells.

Aim The purpose of the current study was to investigate the effects of nephronectin (Npnt) in human dental pulp stem cells (hDPSCs). Methodology Npnt was coated to nontissue culture-treated polystyrene (non-PS) plates. The presence of immobilized protein on the surface was detected by polyclonal rabbit primary anti-Npnt antibody. Then the cell number was counted and compared with PBS-, bovine serum albumin- (BSA-), fish scale type I collagen- (FCOL1-), and human fibronectin- (Fn-) coated wells. Cell proliferation was assessed using CCK-8 assay. Cell morphology was observed under light microscopy and fluorescence microscopy. Lastly, the mRNA expression profiles of integrins, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and mineralization capacity of hDPSCs were investigated by real time RT-PCR and alizarin red staining, respectively. Results Npnt mediates hDPSC adhesion and spreading partially via the Arg-Gly-Asp (RGD) motif. Npnt enhanced the mRNA expression of ITGA1, ITGA4, ITGA7, and ITGB1 on day five. Npnt downregulated DSPP but significantly upregulated BSP mRNA expression at day 28. Further, Npnt and FCOL1 accelerated the matrix mineralization in hDPSCs. Conclusions The current findings implicate that Npnt would be favorable to recruit hDPSCs and conducive to mineralization in hDPSCs. The combination of Npnt with hDPSCs may offer a promising approach for hard tissue regeneration.

Marker-free method for accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features.

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100 nm.

Eosinophils release extracellular DNA traps in response to Aspergillus fumigatus

Eosinophils mediate the immune response in different infectious conditions. The release of extracellular DNA traps (ETs) by leukocytes has been described as an innate immune response mechanism that is relevant in many disorders including fungal diseases. Different stimuli induce the release of human eosinophil ETs (EETs). Aspergillus fumigatus is an opportunistic fungus that may cause eosinophilic allergic bronchopulmonary aspergillosis (ABPA). It has been reported that eosinophils are important to the clearance of A fumigatus in infected mice lungs. However, the immunological mechanisms that underlie the molecular interactions between A fumigatus and eosinophils are poorly understood. Here, we investigated the presence of EETs in the bronchial mucus plugs of patients with ABPA. We also determined whether A fumigatus induced the release of human eosinophil EETs invitro. Mucus samples of patients with ABPA were analyzed by light and confocal fluorescence microscopy. The release of EETs by human blood eosinophils was evaluated using different pharmacological tools and neutralizing antibodies by fluorescence microscopy and a fluorimetric method. We identified abundant nuclear histone-bearing EETs in the bronchial secretions obtained from patients with ABPA. Invitro, we demonstrated that A fumigatus induces the release of EETs through a mechanism independent of reactive oxygen species but associated with eosinophil death, histone citrullination, CD11b, and the Syk tyrosine kinase pathway. EETs lack the killing or fungistatic activities against A fumigatus. Our findings may contribute to the understanding of how eosinophils recognize and act as immune cells in response to A fumigatus, which may lead to novel insights regarding the treatment of patients with ABPA.

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (−195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

Wide-field imaging through scattering media by scattered light fluorescence microscopy

To obtain images through scattering media, scattered light fluorescence (SLF) microscopy that utilizes the optical memory effect has been developed. However, the small field of view (FOV) of SLF microscopy limits its application. In this paper, we have introduced a re-modulation method to achieve wide-field imaging through scattering media by SLF microscopy. In the re-modulation method, to raster scan the focus across the object plane, the incident wavefront is re-modulated via a spatial light modulator (SLM) in the updated phase compensation calculated using the optimized iterative algorithm. Compared with the conventional optical memory effect method, the re-modulation method can greatly increase the FOV of a SLF microscope. With the phase compensation theoretically calculated, the process of updating the phase compensation of a high speed SLM is fast. The re-modulation method does not increase the imaging time. The re-modulation method is, therefore, expected to make SLF microscopy have much wider applications in biology, medicine and physiology.

Initial Stages of Angiogenesis after Acute Experimental Local Venous Outflow Disturbances and Application of Cell Technologies.

The initial stages of angiogenesis in rats after transcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained cell membranes in the projection of ligated femoral vein were studied by fluorescent light and confocal laser microscopy. Large clusters of brightly fluorescing elongated fibroblast-like cells were seen in the paravasal tissue and in the postoperative scar and signs of angiogenesis were noted as soon as in 4 days. The injected cells not only formed new vessels, but also integrated into vessels formed by host cells. Some injected cells were phagocytizied by macrophages and the latter started to fluoresce due to the presence of the membrane dye. These macrophages within the specified period appeared in the regional inguinal lymph nodes where they formed clusters in the lymphoid parenchyma of the cortical substance.

Super-resolution by annular pupil inserted scattered light fluorescence microscopes

Scattered light fluorescence (SLF) microscopy has been developed to take images through scattering media with subwavelength resolution. Here, we investigate the effect of an annular pupil on SLF microscopes via theory and simulations. As opposite to subwavelength resolution achieved by the conventional SLF microscopes, super-resolution can be achieved by annular pupil inserted scattered light fluorescence (API-SLF) microscopes. In fact, the resolution of an API-SLF microscope is usually relevant to one of its component, the scattering medium. By showing the average resolution of API-SLF microscopes that composed of different scattering media is about 0.4 times of the incident wavelength, we demonstrate that an API-SLF microscope with appropriate scattering medium is able to beat the diffraction limit.

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. By working with cells that have been rapidly frozen without the use of chemical fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.

Direct Evidence of Lack of Colocalisation of Fluorescently Labelled Gold Labels Used in Correlative Light Electron Microscopy

Fluorescently labelled nanoparticles are routinely used in Correlative Light Electron Microscopy (CLEM) to combine the capabilities of two separate microscope platforms: fluorescent light microscopy (LM) and electron microscopy (EM). The inherent assumption is that the fluorescent label observed under LM colocalises well with the electron dense nanoparticle observed in EM. Herein we show, by combining single molecule fluorescent imaging with optical detection of the scattering from single gold nanoparticles, that for a commercially produced sample of 10 nm gold nanoparticles tagged to Alexa-633 there is in fact no colocalisation between the fluorescent signatures of Alexa-633 and the scattering associated with the gold nanoparticle. This shows that the attached gold nanoparticle quenches the fluorescent signal by ~95%, or less likely that the complex has dissociated. In either scenario, the observed fluorescent signal in fact arises from a large population of untagged fluorophores; rendering these labels potentially ineffective and misleading to the field.

Effect of calcitonin on cultured schwann cells.

After nerve injury, calcium concentrations in intranerve fibers quickly increase. We have shown that functional recovery of injured nerves correlates with calcium absorption. A slight increase in calcium reduces the number of Schwann cells present. Calcitonin therapy greatly improves regeneration by accelerating calcium absorption. We examined the effect of adding calcitonin to higher concentration calcium media on cultured Schwann cells. The cells, isolated from intact sciatic nerves, were cultured with normal or higher concentration calcium media with or without calcitonin. Schwann cells were incubated with anti-S-100, goat-anti-mouse, and propidium iodide and then viewed through fluorescent light and phase-contrast microscopy for observation and analysis. The cells in each calcitonin-containing medium showed many Schwann cells, however, the cells in the higher concentration calcium media showed fewer and more defective Schwann cells. These results show that calcitonin protects against the harmful effects of excessive calcium encountered in peripheral nerve injury. Muscle Nerve 56: 768-772, 2017.

Characterization of tar-like material and producible oil in Misoa C2 and C3 reservoir sands of the Area 2 South (Ceuta Field, Venezuela)

Here we characterized asphaltic oil occurrences in the Misoa C2 and C3 reservoirs within Area 2 South of the Ceuta Field (Lake Maracaibo Basin, NW Venezuela). To perform the geochemical and petrological characterization of the C2 and C3 sub-units, which cover an interval in which tar-like material was detected, we analyzed core (18) and cutting (123) samples using the Rock-Eval 6 technique in reservoir mode. Samples were studied under fluorescence light and scanning electron microscopy (SEM), showing the presence of two charges of oils and the relationships within silica cements and clay minerals. To evaluate vertical inter-reservoir communication and lateral intra-reservoir continuity, we also studied 12 oil samples from wells in Area 2 South. Biomarkers and isotopic signatures were examined using gas chromatography-mass spectrometry (GC-MS) and isotope ratio mass spectrometry (IRMS). The results suggest that oil and core extracts belongs to a single family derived from two pulses of hydrocarbon generation from the La Luna source rock, which was deposited in a marine reducing environment. The oil samples are a mixture of a paleobiodegraded oil charge and a later fresh, more mature oil recharge. The geochemical analyses also evidence that the formation of heavy oil tar zones can be related to biodegradation and in-reservoir oil mixing, though other formation mechanisms for the tar are also feasible. Finally, the GC fingerprints of the oils are indicative of lateral reservoir continuities across the F-1 and F-2 faults and vertical communication between the Misoa C2 and C3 sub-units in Area 2 South, suggesting that tar occurrences do not act as permeability barriers within the two reservoirs studied.

The role of the pAbp gene encoding the cytoplasmic poly(A)-binding protein in spermatogenesis of Drosophila melanogaster

The Drosophila melanogaster pAbp gene encodes the cytoplasmic poly(A)-binding protein (PABP). Cytoplasmic PABPs participate in the metabolic pathways of the mRNA: translation initiation and termination, cytoplasmic polyadenylation/deadenylation, mRNA stability, mRNA degradation. Despite the extensive biochemical and structural characterization, relatively little is known about the biological roles of PABPs in the processes of cellular development and differentiation. In Drosophila spermatogenesis, posttranscriptional mechanisms of gene regulation play an important role, so cytoplasmic PABP can have significant function in this process. Deletion of PABP leads to embryonic lethality. However, some flies carrying combinations of mutant pAbp alleles survive but display male sterility and show defects in spermatogenesis. It has previously been shown that hypomorphic pAbp mutations cause a number of meiotic defects, abnormalities of Nebenkern formation. These data provide an insight into the effect of pAbp mutation on the individual events of spermatogenesis, but they do not cover the entire process. We studied spermatogenesis in pAbp allele heterozygotes by transmission electron and fluorescent light microscopy. We showed that cellular mitochondria were the primary structural target of the mutation. Abnormal mitochondria were less structured, swollen, had transparent matrix and depleted cristae. Further mutation had a polymorphous effect and induced anomalies in the ultrastructure of mature spermatocytes, defects in Nebenkern formation and division, axial complex formation, shutdown of spermatogenesis during spermatid elongation. Thus our data show a significant role of PABP in structural transformations of male germ cells during entire spermato-genesis.

Morphological Characteristics of a Rat Model of Reexpansion Pulmonary Edema

Objective: Reexpansion pulmonary edema (RPE) is a severe disorder, and its pathophysiology is not well understood. One proposed mechanism for RPE is that chemical substances such as cytokines increase alveolar permeability. Another possible mechanism is that alveolar distention during reexpansion causes physical damage. To test the hypothesis that sudden alveolar distention damages alveolar cellular structure and identify the underlying cause of RPE, we developed and evaluated the morphological characteristics of a rat RPE model. Methods: Lung from a rat model of RPE was observed by using live imaging from intravital fluorescence microscopy with fluorescein isothiocyanate labelled albumin as tracer, light microscopy, and electron microscopy (with and without horseradish peroxidase [HRP] as tracer). Results: Intravital fluorescence microscopy and light microscopy showed that RPE developed almost immediately after lung reexpansion and that blood flow in pulmonary capillaries slowed substantially. In some capillaries, however, blood flow had stopped entirely; in others, anterograde and retrograde flow alternated. Electron microscopy revealed pores in type I pneumocytes and overt fissures in alveolar walls. Electron microscopic observation with HRP revealed that HRP moved from capillaries to the inner surfaces of alveolar epithelia in reexpanded lungs. In addition, diaminobenzidine reaction products from the HRP enzyme reaction were visible in areas with pores. Conclusions: RPE occurred almost immediately after lung reexpansion. Pores developed in type I pneumocytes in alveolar epithelium. These pores, together with overt fissures in alveolar walls, allowed leakage of plasma components into alveoli. These findings appear to be important features of RPE development.

In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography.

The unique property of electron cryotomography (ECT) is its capability to resolve the structure of macromolecular machines in their cellular context. The integration of ECT data with high-resolution structures of purified subcomplexes and live-cell fluorescence light microscopy can generate pseudo-atomic models that lead to a mechanistic understanding across size and time scales. Recent advances in electron detection, sample thinning, data acquisition, and data processing have significantly enhanced the applicability and performance of ECT. Here we describe a detailed workflow for an ECT experiment, including cell culture, vitrification, data acquisition, data reconstruction, tomogram analysis, and subtomogram averaging. This protocol provides an entry point to the technique for students and researchers and indicates the many possible variations arising from specific target properties and the available instrumentation.

Precise tracking of the dynamics of multiple proteins in endocytic events.

Endocytosis is a complex and dynamic process that involves dozens of different proteins to define the site of endocytosis, form a membrane invagination, and pinch off a membrane vesicle into the cytoplasm. Fluorescent light microscopy is a powerful tool to visualize the dynamic behaviors of the proteins taking part in the endocytic process. The resolution of light microscopy is, however, a serious limitation. Here, we detail a fluorescence microscope method that we have developed to visualize the dynamics of the clathrin-mediated endocytic protein machinery in yeast cells. This method is based on subpixel centroid tracking of endocytic proteins. For each endocytic protein, the centroid trajectories obtained from multiple endocytic events are used to compute an average trajectory that describes, at nanometer scale, the assembly and movement of the protein during endocytosis. The average trajectories of the different endocytic proteins are then aligned together in space and time to reconstruct how the different proteins behave relative to each other during the endocytic process.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM.

Staining with two observational methods for the diagnosis of tuberculous meningitis.

Ziehl-Neelsen (Z-N) staining of cerebrospinal fluid (CSF) for acid-fast bacilli (AFB) is the cornerstone of the laboratory diagnosis of tuberculous meningitis (TBM). However, the sensitivity of conventional Z-N staining for the detection of AFB in CSF specimens is suboptimal. The present study aimed to compare the practicality of modified Z-N staining with light microscopy and fluorescence microscopy in the same smear without auramine O. A total of 155 patients with 223 CSF specimens were enrolled and grouped according to the uniform case definition. The smears of each CSF specimen were subjected to modified Z-N staining and then observed using a light microscope under transmitted light and under fluorescence with a green-excitation wavelength in the same microscopic field. The results for different groups, inspection times, and prior to and following treatment were compared. Results indicated that the fuchsin-stained AFB were visible as bright orange-red fluorescing rods under fluorescence, or as red, lightly curved rods under transmitted light. The sensitivity of fluorescence microscopy was 96.2% while that of light microscopy was 84.6%. The positive rate of fluorescence microscopy was 79.2% prior to treatment compared with 61.7% post-treatment. In the same microscopic field, a greater number of AFB were observed using fluorescence compared with transmitted light, and AFB that were not visible under transmitted light were clearly observed under fluorescence. Furthermore, transmitted light and fluorescence could be interchanged directly when equivocal smears were encountered. The combination of modified Z-N staining and fluorescence microscopy without auramine O is sensitive and convenient for the diagnosis of TBM.