Vascular supply of the anterior interventricular epicardial nerves and ventricular Purkinje fibers in the porcine hearts

The aim of this study was to perform a pilot histological and quantitative analysis of the blood vessels accompanying the epicardial nerves (vasa nervorum) in the porcine hearts. Twenty healthy porcine hearts were used in this study. The blood vessels were analyzed by light microscopy using four different staining techniques in transverse sections taken from the upper, middle, and lower segments of the anterior part of the interventricular region and the adjacent parts of the right and left ventricles containing epicardial nerves and the endocardial peripheral parts of the Purkinje fibers. In total, 317 epicardial nerves were detected. The vasa nervorum were present in 75.7% of these nerves. The vasa nervorum resembled arterioles and postcapillary and collecting venules. One hundred and forty nine epicardial nerves were perivascular, located in the adventitia of the anterior interventricular artery and vein. The remaining 168 nerves ran freely through the epicardial interstitium. The presence of the vasa nervorum was not related to topographical location or nerve diameter. Additionally, from a total of 33 analyzed ventricular complexes of Purkinje fibers small blood vessels located in their proximity were identified in only two cases. It can be concluded that the majority of the anterior epicardial nerves of porcine heart possess well-developed vasa nervorum. In contrast, similar blood vessels are rarely present in the vicinity of the Purkinje fibers. The data obtained contribute to a better understanding of the nutrition of the cardiac nerves.     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。     

Contrast inversion in the epifluorescence of cholesterol-phospholipid monolayers

A number of dihydrocholesterol-phospholipid mixtures have been examined using the epifluorescence microscopy of monolayers at the air-water interface. These mixtures form two coexisting liquids. Fluorescence contrast was provided using a variety of different lipid probes. With increasing monolayer pressure, all of the charged probes show contrast inversion at higher dihydrocholesterol concentrations. That is, with increasing pressure the charged probes transfer from one liquid to the other, reversing the fluorescence contrast. A wide variety of phospholipids were studied, and the inversion was seen in all cases. In the inverted state and at the higher dihydrocholesterol concentrations, the immiscibility persists to the highest pressures employed, 30-40 mN/m. The data show that binary dihydrocholesterol-phospholipid mixtures can form three distinct liquids, one of which is interpreted as a phase rich in condensed complex.     

Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster

The genera Azospirillum, Skermanella and Rhodocista form a phylogenetic subgroup within the alfa subclass of Proteobacteria. Based on comparative 16S rRNA sequence analysis a nested set of new oligonucleotide probes was designed. It comprises probes for the whole genus cluster Azospirillum-Skermanella-Rhodocista, for the Azospirilli subcluster I including A. lipoferum, A. doebereinerae, A. largimobile, A. brasilense and A. halopraeferens, for the Azospirilli subcluster II including A. amazonense, A. irakense and the genus Skermanella, for the genus Rhodocista as well as for all Azospirilli species or species cluster. The new probes allow a fast and reliable in situ identification of bacteria belonging to the Azospirillum-Skermanella-Rhodocista-cluster at different phylogenetic levels. The specificity of the new probes was tested with 56 strains of the Azospirillum-Rhodocista-Skermanella-cluster and selected reference cells from other genera by hybridising with the complete probe set. In addition, applications of the fluorescently labelled probes for in situ identification of isolates and for the in situ localisation of A. brasilense on maize roots were demonstrated using confocal laser scanning microscopy.     

钙荧光探剂的研究及其在生命科学中的应用

钙荧光探剂测量活细胞胞浆游离Ｃａ２＋浓度的方法在钙研究中已成为一种越来越重要的技术。特别是由于新的一代荧光探剂的合成和激光共聚焦显微镜的发展,使其应用更加广泛。由于国内使用这种技术的实验室逐渐增多,本文将系统介绍钙荧光探剂的发展、测量原理和方法、新的常用钙荧光探剂的比较及其在生命科学中的应用。     

Comparison and Evaluation of Silver Probe Preparation Techniques for Tip-Enhanced Raman Spectroscopy

In this work, the different procedures for the fabrication of Ag probes for tip-enhanced Raman spectroscopy (TERS) in a top illumination/detection setup are proposed and tested. We focus on technologically simple methods allowing Si tips coated with plasmonic silver nanostructures and bulk metal Ag tips with good shape reproducibility to be produced for atomic force microscopy (AFM) feedback setup. The preparation of Ag TERS probes was based on chemical deposition and vacuum sputtering of Ag on the tips of commercially available Si cantilevers. A straightforward technique for the fabrication of bulk metal Ag probes by the electrochemical etching of Ag microwires was also proposed. Chemically coated, sputtered, and electrochemically etched TERS tips were characterized by scanning electron microscopy (SEM). The produced tips were tested for TERS measurements using graphene oxide (GO) as the target analyte in a top illumination setup. A comparative analysis of enhancement factors (EF) for the different types of tips (probes) is presented in this work.     

Confocal laser scanning microscopy as an analytical tool in chromatographic research

In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.     

In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.     

Microvascularization of the spleen in larval and adult Xenopus laevis: Histomorphology and scanning electron microscopy of vascular corrosion casts

Microvascular anatomy and histomorphology of larval and adult spleens of the Clawed Toad, Xenopus laevis were studied by light microscopy of paraplast embedded serial tissue sections and scanning electron microscopy (SEM) of vascular corrosion casts (VCCs). Histology showed i) that white and red pulp are present at the onset of metamorphic climax (stage 57) and ii) that splenic vessels penetrated deeply into the splenic parenchyma at the height of metamorphic climax (stage 64). Scanning electron microscopy of VCCs demonstrated gross arterial supply and venous drainage, splenic microvascular patterns as well as the structure of the interstitial (extravasal) spaces representing the “open circulation routes.” These spaces identified themselves as interconnected resin masses of two distinct forms, namely “broccoli‐shaped” forms and highly interconnected small resin structures. Arterial and venous trees were clearly identified, as were transitions from capillaries to interstitial spaces and from interstitial spaces to pulp venules. Venous sinuses were not diagnosed (nonsinusal spleen). The splenic circulation in Xenopus laevis is “open.” It is hypothesized that red blood cells circulate via splenic artery, central arteries, penicillar arteries, and red pulp capillaries primarily via “broccoli‐shaped” interstitial spaces, pulp venules and veins into subcapsular veins to splenic veins while lymphocytes circulate also via the interstitial spaces represented by the highly interconnected small resin structures in vascular corrosion casts. In physiological terms, the former most likely represent the fast route for blood circulation, while the latter represent the slow route. J. Morphol. 277:1559–1569, 2016. © 2016 Wiley Periodicals, Inc.     

Microscale Analyses of the Formation and Nature of Microbial Biofilm Communities in River Systems

The review covers aspects of biofilm cultivation, laser scanning microscopy, molecular probes and digital image analyses. This is accomplished through an overview of selected studies which illustrate the application of the microscale approach and laser microscopy techniques to the study of river biofilms and the results obtained.     

Fabrication and operation of GRIN probes for in vivo fluorescence cellular imaging of internal organs in small animals

Intravital fluorescence microscopy has emerged as a powerful technique to visualize cellular processes in vivo. However, owing to their size, the objective lenses required have limited physical accessibility to various tissue sites in the internal organs of small animals. The use of small-diameter probes using graded-index (GRIN) lenses expands the capabilities of conventional intravital microscopes to minimally invasive imaging of internal organs. In this protocol, we describe the detailed steps for the fabrication of front- and side-view GRIN probes and the integration and operation of the probes in a confocal microscope to enable visualization of fluorescent cells and microvasculature in various mouse organs. Some experience in building an optical setup is required to complete the protocol. We also present longitudinal imaging of immune cells in renal allografts and tumor development in the colon. Fabrication and integration can be completed in 5-7 h, and a typical in vivo imaging session takes 1-2 h.     

Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy

We describe a method based on fluorescence in situ hybridisation (FISH) that allows the identification of individual cells by electron microscopy. We hybridised universal and specific fluorescein-labelled oligonucleotide probes to the ribosomal RNA of prokaryotic microorganisms in heterogeneous cell mixtures. We then used antibodies against fluorescein coupled to sub-nanometer gold particles to label the hybridised probes in the ribosome. After increasing the diameter of the metal particles by silver enhancement, the specific gold-silver signal was visualised by optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is the first time that SEM is applied to the detection of gold nanoparticles hybridised to an intracellular target, such as the ribosome. The possibility to couple phylogenetic identification by FISH to cell surface and ultrastructure observation at electron microscopy resolution has promising potential applications in microbial ecology.     

Plant cell biology in the new millennium: new tools and new insights

The highly regulated structural components of the plant cell form the basis of its function. It is becoming increasingly recognized that cellular components are ordered into regulatory units ranging from the multienzyme complexes that allow metabolic channeling during primary metabolism to the "transducon" complexes of signal transduction elements that allow for the highly efficient transfer of information within the cell. Against this structural background the highly dynamic processes regulating cell function are played out. Recent technological advances in three areas have driven our understanding of the complexities of the structural and functional dynamics of the plant cell. First, microscope and digital camera technology has seen not only improvements in the resolution of the optics and sensitivity of detectors, but also the development of novel microscopy applications such as confocal and multiphoton microscopy. These technologies are allowing cell biologists to image the dynamics of living cells with unparalleled three-dimensional resolution. The second advance has been in the availability of increasingly powerful and affordable computers. The computer control/analysis required for many of the new microscopy techniques was simply unavailable until recently. Third, there have been dramatic advances in the available probes to use with these new microscopy approaches. Thus the plant cell biologist now has available a vast array of fluorescent probes that will report cell parameters as diverse as the pH of the cytosol, the oxygen level in a tissue, or the dynamics of the cytoskeleton. The combination of these new approaches has led to an increasingly detailed picture of how plant cells regulate their activities.     

Usefulness of genus-specific PCR and Southern blot species-specific hybridization for the detection of imported malaria cases in Italy

A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.     

Utilization of fluorescent probes for the quantification and identification of subcellular proteomes and biological processes regulated by lipid peroxidation products

Oxidative modifications to cellular proteins are critical in mediating redox-sensitive processes such as autophagy, the antioxidant response, and apoptosis. The proteins that become modified by reactive species are often compartmentalized to specific organelles or regions of the cell. Here, we detail protocols for identifying the subcellular protein targets of lipid oxidation and for linking protein modifications with biological responses such as autophagy. Fluorophores such as BODIPY-labeled arachidonic acid or BODIPY-conjugated electrophiles can be paired with organelle-specific probes to identify specific biological processes and signaling pathways activated in response to oxidative stress. In particular, we demonstrate “negative” and “positive” labeling methods using BODIPY-tagged reagents for examining oxidative modifications to protein nucleophiles. The protocol describes the use of these probes in slot immunoblotting, quantitative Western blotting, in-gel fluorescence, and confocal microscopy techniques. In particular, the use of the BODIPY fluorophore with organelle- or biological process-specific dyes and chromophores is highlighted. These methods can be used in multiple cell types as well as isolated organelles to interrogate the role of oxidative modifications in regulating biological responses to oxidative stress.     

Structural and optical properties of dysprosium-doped hydroxyapatite nanoparticles and the use as a bioimaging probe in human cells

In this work, the hydroxyapatite nanoparticles doped with trivalent dysprosium ions were synthesized by a co-precipitation method. The characterization techniques such as X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX) were carried out to determine the crystalline and structural properties. The Rietveld structural refinement of the XRD patterns confirmed the purity of the phase formation of the synthesized nanoparticles. The photoluminescence emission spectra exhibited intense emissions in the blue region at 450 nm and 476 nm along with less intense yellow emission at 573 nm which can be attributed to the magnetic dipole and electric dipole transitions of dysprosium respectively. In order to evaluate the colour tunability of the emitted light CIE chromaticity coordinate values were calculated. The intense blue emissions from the synthesized sample were found to be favourable for bioimaging. The images obtained from the fluorescence microscopy revealed that the dysprosium-doped hydroxyapatite nanoparticles are potential bioimaging probes in human cells.     

Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms.

The population architecture of sulfidogenic biofilms established in anaerobic fixed-bed bioreactors was characterized by selective polymerase chain reaction amplification and fluorescence microscopy. A region of the 16S rRNA common to resident sulfate-reducing bacteria was selectively amplified by the polymerase chain reaction. Sequences of amplification products, with reference to a collection of 16S rRNA sequences representing most characterized sulfate-reducing bacteria, were used to design both general and specific hybridization probes. Fluorescent versions of these probes were used in combination with fluorescence microscopy to visualize specific sulfate-reducing bacterial populations within developing and established biofilms.     

Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms.

The population architecture of sulfidogenic biofilms established in anaerobic fixed-bed bioreactors was characterized by selective polymerase chain reaction amplification and fluorescence microscopy. A region of the 16S rRNA common to resident sulfate-reducing bacteria was selectively amplified by the polymerase chain reaction. Sequences of amplification products, with reference to a collection of 16S rRNA sequences representing most characterized sulfate-reducing bacteria, were used to design both general and specific hybridization probes. Fluorescent versions of these probes were used in combination with fluorescence microscopy to visualize specific sulfate-reducing bacterial populations within developing and established biofilms.     

Application of rRNA-based probes for observing marine nanoplanktonic protists.

The use of small-subunit rRNA-based oligonucleotides as probes for detecting marine nanoplanktonic protists was examined with a ciliate (an Uronema sp.), a flagellate (a Cafeteria sp.), and mixed assemblages of protists from enrichment cultures and natural seawater samples. Flow cytometry and epifluorescence microscopy analyses demonstrated that hybridizations employing fluorescein-labeled, eukaryote-specific probes intensely stained logarithmically growing protists, whereas these same protist strains in late stationary growth were barely detectable. The fluorescence intensity due to probe binding was significantly enhanced by the use of probes end labeled with biotin, which were detected by fluorescein-labeled avidin. The degree of signal amplification ranged from two- to fivefold for cultured protists in both logarithmic and stationary growth phases. Mixed assemblages of heterotrophic protists from enrichment cultures were also intensely labeled by rRNA-targeted oligonucleotide probes by the biotin-avidin detection system. Protists in late stationary growth phase and natural assemblages of protists that were otherwise undetectable when hybridized with fluorescein-labeled probes were easily visualized by this approach. In the latter samples, hybridization with multiple, biotin-labeled probes was necessary for detection of naturally occurring marine protists by epifluorescence microscopy. The signal amplification obtained with the biotin-avidin system should increase the utility of rRNA-targeted probes for identifying protists and facilitate characterization of the population structure and distribution of protists in aquatic environments.     

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores.Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.