Morphology of the cochlear nerve in Sprague-Dawley and brown Norway rats

Cochlear nerve morphology was studied in young adult albino (Sprague-Dawley) and pigmented (Brown Norway) rats. Analysis of the material included counts of normal and degenerating fibers and of glial cell nuclei, and measurements of vascularity and of the nerves' cross-sectional areas. The median number of normal fibers in the Sprague-Dawley rats was 21,216, and, in the Brown Norway rats, it was 20,186. There were no statistically significant differences between the two strains in numbers of normal fibers, degenerating myelin sheaths, or glial cell nuclei, or in the cross-sectional areas of the nerves. The area density of blood vessels was significantly higher in nerves from the Sprague-Dawley rats. The median area density in that strain was 0.0149, while in the Brown Norway rats the median area density was 0.0105.     

Imaging the structure and organization of mouse cerebellum and brain stem with second harmonic generation microscopy

To visualize the structure and organization of the brain is a fundamental requirement in the research of neuroscience. Here, combining with two-photon excitation fluorescence microscopy and transgenetic mouse GAD67,we demonstrate a custom-built second harmonic generation(SHG) microscope to discriminate brain layers and sub regions in the cerebellum and brain stem slices with cellular resolution. In particular, the cell densities of neurons in different brain layers are extracted due to the cell soma appearing as dark shadow on an SHG image.Further, the axon initial segments of the Purkinje cell are easily recognized without labeling, which would be useful for guiding micropipettes for electrophysiology.     

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy.     

The assessment of viability in isolated rat hepatocytes subjected to cold or subzero non-freezing preservation protocols using a propidium iodide modified test

A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence--Fd) and after (total fluorescence--Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.     

Relationship between adsorbed fibronectin and cell adhesion on a honeycomb-patterned film

Substratum surface morphology plays a vital roles in cellular behavior. Here, we characterized adsorption of fibronectin (Fn) as a typical cell adhesion protein onto honeycomb-patterned films made of poly(ε-caprolactone) (PCL) by using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). In order to determine how cells adhere to a honeycomb-patterned film, focal adhesion of cardiac myocytes (CMYs) and endothelial cells (ECs) on the films were studied by using fluorescence labeling of vinculin. Fn adsorbs around the pore edges to form ring-shaped structures. CMYs and ECs adhere onto the honeycomb-patterned films at focal contact points localized around pore edges distributed over the entire cellular surface. The focal contact points on the honeycomb-patterned films correspond well with the adsorption sites of Fn. We suggest that the cell response to honeycomb-patterned films is associated with the adsorption pattern of Fn on the film.     

Genetic analysis of posterior medial barrel subfield (PMBSF) size in somatosensory cortex (SI) in recombinant inbred strains of mice

Background  

Quantitative trait locus (QTL) mapping is an important tool for identifying potential candidate genes linked to complex traits. QTL mapping has been used to identify genes associated with cytoarchitecture, cell number, brain size, and brain volume. Previously, QTL mapping was utilized to examine variation of barrel field size in the somatosensory cortex in a limited number of recombinant inbred (RI) strains of mice. In order to further elucidate the underlying natural variation in mouse primary somatosensory cortex, we measured the size of the posterior medial barrel subfield (PMBSF), associated with the representation of the large mystacial vibrissae, in an expanded sample set that included 42 BXD RI strains, two parental strains (C57BL/6J and DBA/2J), and one F1 strain (B6D2F1). Cytochrome oxidase labeling was used to visualize barrels within the PMBSF.     

CSF from Parkinson disease Patients Differentially Affects Cultured Microglia and Astrocytes

Background  

Excessive and abnormal accumulation of alpha-synuclein (α-synuclein) is a factor contributing to pathogenic cell death in Parkinson's disease. The purpose of this study, based on earlier observations of Parkinson's disease cerebrospinal fluid (PD-CSF) initiated cell death, was to determine the effects of CSF from PD patients on the functionally different microglia and astrocyte glial cell lines. Microglia cells from human glioblastoma and astrocytes from fetal brain tissue were cultured, grown to confluence, treated with fixed concentrations of PD-CSF, non-PD disease control CSF, or control no-CSF medium, then photographed and fluorescently probed for α-synuclein content by deconvolution fluorescence microscopy. Outcome measures included manually counted cell growth patterns from day 1-8; α-synuclein density and distribution by antibody tagged 3D model stacked deconvoluted fluorescent imaging.     

Proliferation dynamics of germinative zone cells in the intact and excitotoxically lesioned postnatal rat brain

Background  

The forebrain subventricular zone (SVZ)-olfactory bulb pathway and hippocampal subgranular zone (SGZ) generate neurons into adulthood in the mammalian brain. Neurogenesis increases after injury to the adult brain, but few studies examine the effect of injury on neural and glial precursors in the postnatal brain. To characterize the spatio-temporal dynamics of cell proliferation in the germinative zones, this study utilized a model of postnatal damage induced by NMDA injection in the right sensorimotor cortex at postnatal day 9.     

聚乙烯吡咯烷酮修饰的NaYF4∶Yb3+/Er3+上转换 发光纳米粒子的制备及其生物兼容性

以聚乙烯吡咯烷酮(PVP)为表面活性剂,利用溶剂热法合成了NaYF₄∶20%Yb³⁺, 3%Er³⁺(摩尔分数)的上转换发光纳米粒子。利用扫描电子显微镜对粒子的形貌进行了表征,结果表明纳米颗粒的尺寸在30~40 nm,分布比较均匀。在980 nm红外光的激发下,样品能够发出肉眼可见的明亮的黄色上转换荧光。样品可以较好地分散在水溶液中形成透明澄清的溶液。利用MTT实验测量了不同给药浓度下NaYF₄纳米粒子是否对HeLa细胞具有生物毒性。结果表明:PVP修饰的NaYF₄∶Yb³⁺/Er³⁺上转换发光纳米粒子具有较好的生物兼容性,对HeLa细胞无生物毒性,在生物荧光标记领域具有潜在的应用价值。     

GFP immunogold staining, from light to electron microscopy, in mammalian cells

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one hand it allows adequate fixation for EM analysis maintaining GFP antigenicity, on the other hand it also enables the epoxy resins inclusion after immunogold staining. Both of them help to preserve better the ultrastructure. However GFP immunogold staining presents some drawbacks, such as the progressive decrease in immunogold labeling with tissue depth. Special attention must be taken when using GFP-tagged protein, since the fusion could interfere with their localization and function. In this review we provide a detailed protocol of the GFP immunogold staining, their main applications for EM and possible troubles.     

Immuno-localization of Fas and FasL in rat hepatic endothelial cells: influence of different fixation protocols

Immunocytochemistry has been widely used to localize molecules involved in apoptosis. In this short report, we describe with the aid of confocal laser scanning microscopy the immunolocalization of Fas and FasL on liver sinusoidal endothelial cells, and show how the localization of these two molecules differ when the cells are fixed with different fixation protocols. Methanol fixation shows diffuse staining of Fas and FasL in the cytoplasm, as well as in the nucleus. In contrast, paraformaldehyde fixation reveals the presence of Fas and FasL polarized at one side of the cell and only in the cytoplasm. After fixation with a combination of paraformaldehyde and glutaraldehyde the polarization is still present although the fluorescence is concentrated and located as bright dots in the cytoplasm. In conclusion, paraformaldehyde preserves the (nuclear) membrane-associated structures better then methanol and results in a more accurate localization of Fas and FasL. Understanding the different outcome of these common used fixation protocols will assist investigators to select the most suitable method for visualizing membrane-bound Fas and FasL.     

Detection of Volume Changes in Calcein-Stained Cells Using Confocal Microscopy

Calcein is an intracellular fluorescent probe that has been used as an indicator of cell volume in several previous studies. These studies have reported two different fluorescence responses depending on the optical setup used to collect the data: wide-field microscopy has resulted in a decrease in fluorescence upon cell shrinkage, whereas confocal microscopy has been shown to yield the opposite result. In this short communication, we have investigated the effect of optical setup on detection of cell volume changes in calcein-stained endothelial cells. A confocal microscope was used to collect the fluorescence data, and the pinhole diameter was varied in order to examine the effects of optical section thickness on fluorescence response. For large pinhole diameters – which correspond to relatively thick optical sections – fluorescence intensity decreased when cells were induced to shrink. In contrast, for small pinhole diameters the fluorescence intensity increased with cell shrinkage. The transition between these two types of fluorescence responses occurred when using a pinhole diameter of 285 μm, which corresponds with an optical section thickness slightly less than the height of the cells. Our results have implications for the design and interpretation of experiments involving the use of calcein as a cell volume indicator.     

Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals - Quantum Dots (QDs) - have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties - wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.     

In vivo dynamic turnover of cerebral 13C isotopomers from [U-13C]glucose

An INEPT-based (13)C MRS method and a cost-effective and widely available 11.7 Tesla 89-mm bore vertical magnet were used to detect dynamic (13)C isotopomer turnover from intravenously infused [U-(13)C]glucose in a 211 microL voxel located in the adult rat brain. The INEPT-based (1)H-->(13)C polarization transfer method is mostly adiabatic and therefore minimizes signal loss due to B(1) inhomogeneity of the surface coils used. High quality and reproducible data were acquired as a result of combined use of outer volume suppression, ISIS, and the single-shot three-dimensional localization scheme built in the INEPT pulse sequence. Isotopomer patterns of both glutamate C4 at 34.00 ppm and glutamine C4 at 31.38 ppm are dominated first by a doublet originated from labeling at C4 and C5 but not at C3 (with (1)J(C4C5) = 51 Hz) and then by a quartet originated from labeling at C3, C4, and C5 (with (1)J(C3C4) = 35 Hz). A lag in the transition of glutamine C4 pattern from doublet-dominance to quartet dominance as compared to glutamate C4 was observed, which provides an independent verification of the precursor-product relationship between neuronal glutamate and glial glutamine and a significant intercompartmental cerebral glutamate-glutamine cycle between neurons and glial cells.     

水溶性NaYF_4∶Yb/Tm纳米粒子的制备及其上转换发光性质

利用溶剂热法合成了NaYF4:20%Yb,0.5%Tm上转换发光纳米粒子(UCNPs),用扫描电子显微镜、X射线衍射分析、发光光谱测量等手段对水溶性纳米颗粒进行了形貌和发光性质表征。结果表明,UCNPs是纯立方相的NaYF4,尺寸均匀分布在30nm左右。在980nm红外光的激发下,UCNPs能够发出肉眼可见的明亮的蓝紫色光。发射光谱中最强发射峰在479nm,来源于Tm3+离子的1G4→3H6发射,并且给出了UCNPs的上转换发光机制。利用聚乙烯吡咯烷酮(PVP)作为表面活性剂所制备的上转换发光纳米颗粒具有良好的水溶性,尺寸较小,在生物荧光标记领域具有潜在的应用价值。     

Fluorescent Function-Spacer-Lipid Construct Labelling Allows for Real-Time in Vivo Imaging of Cell Migration and Behaviour in Zebrafish (Danio Rerio)

Real-time in vivo imaging of cell migration and behavior has advanced our understanding of physiological processes in situ, especially in the field of immunology. We carried out the transplantation of a mixed population of blood cells from adult zebrafish (Danio rerio) to 2?day old embryos. The blood cells were treated ex vivo with Function-Spacer-Lipid constructs (FSL) incorporating either fluorescein or Atto488 fluorophores (FSL-FLRO4-I or -II). Excellent labeling efficiency was demonstrated by epifluorescence microscopy and FACScan analysis. Real-time video imaging of the recipient fish showed that the functionality of these cells was retained and not affected by the labeling. The usefulness of FSL-FLRO4-I as a contrast agent in microangiography was explored. Overall, we found both FSL-FLRO4-I and-II promising labeling dyes for real-time in vivo imaging in zebrafish.     

Evaluation of Fluorescent Phosphatidylserine Substrates for the Aminophospholipid Flippase in Mammalian Cells

A series of fluorescent phosphatidylserine and phosphatidylcholine derivatives were prepared and evaluated by cell microscopy for ability to translocate across mammalian plasma membranes via the putative aminophospholipid flippase. Phosphatidylserine derivatives, with either a neutral 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or a coumarin fluorophore appended to the 2-acyl chain, entered the cytosol of all three cell lines tested and control experiments showed that the translocation was due to flippase activity. In contrast, a phosphatidylserine conjugate containing a charged and polar carboxyfluorescein was not translocated and remained in the cell plasma membrane. The phosphatidylserine-coumarin derivative exhibits bright fluorescence and higher photostability than the NBD analogues, and thus is a promising new fluorescent probe for extended-imaging studies of flippase action in living cells using laser confocal microscopes.     

Molecular third-harmonic-generation microscopy through resonance enhancement with absorbing dye

This paper reports a facile and effortless method to realize three-dimensional (3D) molecular third-harmonic-generation (THG) microscopy through the technique of resonance enhancement with absorbing dye. Hematoxylin, a popular absorbing stain, is applied as an example to verify the multiphoton resonant enhancement based on the 1230 nm excitation light and can selectively enhance THG yield at cell nuclear sites in the studied specimens, serving as a cell nucleus contrast agent. It is concluded that combining THG microscopy with the mature staining technique can readily achieve 3D molecular imaging without using fluorescence.