High-resolution miniprobe-based confocal microscopy in combination with video mosaicing (with video)

BACKGROUND: A new portable confocal laser microscopy system has recently been developed. OBJECTIVE: Our purpose was to evaluate the feasibility of performing real-time microscopic imaging with a prototype of a new high-resolution miniprobe in conjunction with a video mosaicing algorithm. DESIGN: Feasibility study. SETTING: Tertiary referral center at a large university hospital. PATIENTS: Seven patients referred for endoscopy for various indications. MAIN OUTCOME MEASUREMENTS: High-resolution laser microscopy of the upper and lower GI tract was performed with standard endoscopes. Seven to 10 mL of 1% fluorescein was injected intravenously a few seconds before the procedure. No additional preparation was required. The prototype used has a lateral resolution of 1.2 microm and an axial resolution of 3 mum with a total field of 240 x 200 microm. From all stored video sequences a video mosaicing algorithm was used to combine the successive individual images, cancel motion artifacts, and reconstitute panoramas of the tissues. RESULTS: Cell-to-cell borders, single cell structures, and mucosal inflammation was readily detectable. By the use of the mosaicing algorithm, the image area could be increased 2- to 4-fold, and image definition could be further enhanced to allow finer detail visualization. LIMITATIONS: Low number of patients, early feasibility study. CONCLUSIONS: Our preliminary data show that high-resolution miniprobe-based confocal fluorescence microscopy in conjunction with video mosaicing has the potential to provide images similar to standard histopathologic studies. Dynamic images with a smaller field of view can be combined to reconstruct still images of high resolution covering a fairly large area.     

Characterization of tissue autofluorescence in Barrett''s esophagus by confocal fluorescence microscopy

High grade dysplasia and early cancer in Barrett's esophagus can be distinguished in vivo by endoscopic autofluorescence point spectroscopy and imaging from non-dysplastic Barrett's mucosa. We used confocal fluorescence microscopy for ex vivo comparison of autofluorescence in non-dysplastic and dysplastic Barrett's esophagus. Unstained frozen sections were obtained from snap-frozen Barrett's esophagus biopsy samples and scanned with confocal fluorescence microscopy (458 nm excitation; 505-550 nm [green] and > 560 nm [red] emission). Digital micrographs were taken from areas with homogenous and specific histopathology. Visual inspection and statistical analysis were used to evaluate the image datasets. Dysplastic and non-dysplastic Barrett's esophagus epithelia fluoresced mainly in the green spectrum and the main sources of autofluorescence were the cytoplasm and lamina propria. High-grade dysplasia was differentiated from non-dysplastic Barrett's esophagus by microstructural tissue changes. However, there were no specific changes in either the locations or average intensities of intrinsic green and red autofluorescence at the epithelial level that could differentiate between dysplastic and non-dysplastic Barrett's esophagus epithelia, ex vivo. Detectable differences in autofluorescence between BE and dysplasia/cancer in vivo are probably not caused by specific changes in epithelial fluorophores but are likely due to other inherent changes (e.g. mucosal thickening and increased microvascularity) attenuating autofluorescence from the collagen-rich submucosa. Furthermore, confocal fluorescence microscopy provides 'histology-like' imaging of Barrett's tissues and may offer a unique opportunity to exploit microstructural tissue changes occurring during neoplastic transformation for in vivo detection of high-grade dysplasia in Barrett's patients using newly developed confocal fluorescence microendoscopy devices.     

Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging

Fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS), and photon counting histograms (PCH) are fluctuation methods that emerged recently as potentially useful tools for obtaining parameters of molecular dynamics, interactions, and oligomerization in vivo. Here, we report the successful implementation of FCS, FCCS, and PCH in live yeast cells using fluorescent protein-tagged proteins expressed from their native chromosomal loci, examining cytosolic dynamics and interactions among components of the mitogen activated protein kinase (MAPK) cascade, a widely occurring signaling motif, in response to mating pheromone. FCS analysis detailed the diffusion characteristics and mobile concentrations of MAPK proteins. FCCS analysis using EGFP and mCherry-tagged protein pairs observed the interactions of Ste7 (MAPK kinase) with the MAPKs, Fus3 or Kss1, and of the scaffold protein, Ste5, with Ste7 and Ste11 (MAPK kinase kinase) in the cytosol, providing in vivo constants of their binding equilibrium. The interaction of Ste5 with Fus3 in the cytosol was below the limit of detection, suggesting a weak interaction, if it exists, with K(d) >400-500 nM. Using PCH, we show that cytosolic Ste5 were mostly monomers. Artificial dimerization of Ste5, as confirmed by PCH, using a dimerizing tag, stimulated the interaction between Ste5 and Fus3. Native Ste5 was found to bind Fus3 preferentially at the cortex in pheromone-treated cells, as detected by fluorescence resonance energy transfer (FRET). These results provide a quantitative spatial map of MAPK complexes in vivo and directly support the model that membrane association and regulation of the Ste5 scaffold are critical steps in MAPK activation.     

Ultrasensitive detection of waste products in water using fluorescence emission cavity-enhanced spectroscopy

Clean water is paramount to human health. In this article, we present a technique for detection of trace amounts of human or animal waste products in water using fluorescence emission cavity-enhanced spectroscopy. The detection of femtomolar concentrations of urobilin, a metabolic byproduct of heme metabolism that is excreted in both human and animal waste in water, was achieved through the use of an integrating cavity. This technique could allow for real-time assessment of water quality without the need for expensive laboratory equipment.It is axiomatic that the quality of water is essential for human health (1). The increasing worldwide contamination of freshwater systems with thousands of industrial and natural chemical compounds is one of the key environmental problems facing humanity today, where pathogens in water cause more than 2 million deaths annually (2). With more than one-third of the accessible and renewable freshwater used for industrial, agricultural, and domestic applications, pollution from these activities leaves water sources contaminated with numerous synthetic and geogenic compounds (2, 3). In addition, natural disasters can result in large-scale disruptions of infrastructure, resulting in compromised water quality. Diarrheal disease caused from such disasters may be a major contributor to overall morbidity and mortality rates (4). Thus, the cleanliness and safety of public water sources has prompted researchers to look for rapid and sensitive indicators of water quality. Whereas most water filtering systems are quite efficient in removing large-size contaminants, smaller particles frequently pass through. These contaminants are often poorly soluble in water, thus, present in quantities of less than 1 nM. Here, we demonstrate femtomolar detection of urobilin, a biomarker found in human and animal waste in water.Modern analytical tools have become extremely efficient in the detection and analysis of chemical compounds. For example, liquid chromatography coupled with detection by tandem mass spectrometry has been commonly used for detection of trace pharmaceuticals and other wastewater-derived micropollutants (5). Although such methods are very powerful in identifying trace pollutants, cost prohibits their widespread use by environmental researchers and, most importantly, prevents real-time analysis of water quality (6). Other techniques using bench top gas chromatography–mass spectrometry have also been demonstrated as viable methods for detection of basic pharmaceuticals with reduced cost (7). Despite this, these methods are still cost prohibitive, can hardly be used in field studies, and are unlikely to ever be used for real-time quality control.In addition to pharmaceutical and other synthetic pollutants such as pesticides, animal and human waste (i.e., feces, urine) is an enormous source of water contamination that can be found in both recreational and source waters. These discarded products, when released into water, can carry a variety of diseases such as polio, typhoid, and cholera (8). In extreme cases pollution of an ecosystem can result in environmental crises, such as devastation to the aquatic population, red-tide blooms, as well as beach closings. Molecular methods based on polymerase chain reactions are commonly used to monitor viral, bacterial, and protozoan pathogens in wastewater (9). Microbiological indicators such as fecal coliforms, Escherichia coli and Etherococci, are the indicators most commonly used to analyze and evaluate the level of fecal contamination. However, the suitability of these indicators has been questioned (10), and it takes a substantial time from the extraction of water sample for analysis to the moment when the results are ready.An alternative indicator that has been shown to be helpful in detection of waste in water supplies is urobilin (11). Urobilin is one of the final byproducts of hemoglobin metabolism, and is excreted in both the urine and feces of many mammals, including humans and common livestock (cows, horses, and pigs) (12). In addition, as urobilin can be indicative of disease such as hepatic dysfunction, or jaundice, an ultrasensitive technique for detection and quantification of this biomarker in solution has both diagnostic and environmental applications.Urobilin detection in solution has previously been demonstrated using the formation of a phosphor group from the combination of urobilins and zinc ions (13). Normal heme catabolism results in the production of bilirubin, a red product, which is then broken down into two end products, stercobilin, the bile pigment found in fecal material, and urobilin, the yellow pigment found in urine. Both urobilin and stercobilin have been shown to be viable biomarkers for detection of fecal pollution levels in rivers (14).Fluorescent detection of urobilin in urine has been demonstrated based on Schlesinger’s reaction in which an urobilinogen–zinc chelation complex exhibits a characteristic green fluorescence when excited by blue light (15). Methods for detection of urobilinoids using high-performance liquid chromatography with a reversed-phase column and an ultraviolet detector have also been presented (16); however, the initial sensitivity of this method proved insufficient for clinical analysis. Miyabara and coworkers reported an increase in detection sensitivity of this methodology, but only to detection levels of 1.5 nM (13), where efficient excitation and collection of the fluorescent signal remained the limiting factor. Traditional epiillumination fluorescence spectroscopy systems use an objective lens to focus excitation light into the sample and collect the fluorescence emission. In such a configuration, the signal generated is limited to the focal volume of the optics. In addition, the generated signal is diffusive in nature; only a small fraction of the total emitted light is collected. Because only a small volume of a sample can be probed at any given time with such a configuration, detection of subnanomolar concentrations remains difficult as these measurements are akin to single molecule detection. Thus, a method that could allow for probing a larger volume of a sample while also providing means for collecting more of the fluorescence emission could greatly enhance the ability to detect subnanomolar concentrations of urobilin.     

Conformational changes of calmodulin upon Ca2+ binding studied with a microfluidic mixer

A microfluidic mixer is applied to study the kinetics of calmodulin conformational changes upon Ca2+ binding. The device facilitates rapid, uniform mixing by decoupling hydrodynamic focusing from diffusive mixing and accesses time scales of tens of microseconds. The mixer is used in conjunction with multiphoton microscopy to examine the fast Ca2+-induced transitions of acrylodan-labeled calmodulin. We find that the kinetic rates of the conformational changes in two homologous globular domains differ by more than an order of magnitude. The characteristic time constants are approximately 490 micros for the transitions in the C-terminal domain and approximately 20 ms for those in the N-terminal domain of the protein. We discuss possible mechanisms for the two distinct events and the biological role of the stable intermediate, half-saturated calmodulin.     

Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study

Summary Recent muscle biopsy studies have shown a relation between intramuscular lipid content and insulin resistance. The aim of this study was to test this relation in humans by using a novel proton nuclear magnetic resonance (¹H NMR) spectroscopy technique, which enables non-invasive and rapid ( ∼ 45 min) determination of intramyocellular lipid (IMCL) content. Normal weight non-diabetic adults (n = 23, age 29 ± 2 years, BMI = 24.1 ± 0.5 kg/m²) were studied using cross-sectional analysis. Insulin sensitivity was assessed by a 2-h hyperinsulinaemic ( ∼ 450 pmol/l)-euglycaemic ( ∼ 5 mmol/l) clamp test. Intramyocellular lipid concentrations were determined by using localized ¹H NMR spectroscopy of soleus muscle. Simple linear regression analysis showed an inverse correlation (r = –0.692, p = 0.0017) between intramyocellular lipid content and M-value (100–120 min of clamp) as well as between fasting plasma non-esterified fatty acid concentration and M-value (r = –0.54, p= 0.0267). Intramyocellular lipid content was not related to BMI, age and fasting plasma concentrations of triglycerides, non-esterified fatty acids, glucose or insulin. These results show that intramyocellular lipid concentration, as assessed non invasively by localized ¹H NMR spectroscopy, is a good indicator of whole body insulin sensitivity in non-diabetic, non-obese humans. [Diabetologia (1999) 42: 113–116] Received: 3 September 1998 and in revised form: 11 November 1998     

Antibodies to myelin basic protein,myelin oligodendrocytes peptides,alpha-beta-crystallin,lymphocyte activation and cytokine production in patients with multiple sclerosis

OBJECTIVE: To measure neurone-specific humoral and cellular immune parameters in MRI-positive patients with multiple sclerosis (MS). BACKGROUND: It has been postulated from animal models for MS and in situ evidence in MS patients that antibodies, activated T cells and proinflammatory cytokines are involved in the destruction of myelin sheaths and loss of oligodendrocytes in active areas. SUBJECTS AND METHODS: Blood samples were obtained from 20 healthy control subjects and 20 patients with abnormal MRI and clinical diagnosis of MS. Sera were tested for levels of IgG, IgM and IgA against myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) peptides, and a small heat-shock protein, alpha-beta-crystallin. Lymphocytes were isolated and cultured in the presence or absence of MBP, MOG peptides and alpha-beta-crystallin, measured for stimulated T cells, cytokine production and compared with controls. RESULTS: Patients with MS showed the highest levels of IgG, IgM or IgA antibodies against one or all three tested antigens. Moreover, in the presence of MBP, MOG peptides or alpha-beta-crystallin, a significant percent- age of lymphocytes from MS patients underwent blast transformation, which resulted in high levels of interferon gamma (IFN-gamma), tumour necrosis factor alpha (TNF-alpha) and tumour necrosis factor beta (TNF-beta) production. Sensitivity of these assays was 60-80% and specificity, 65-70%. CONCLUSIONS: Detection of antibodies against MBP, MOG peptides, alpha-beta-crystallin, lymphocyte stimulation and production of proinflammatory cytokines in response to these antigens could be used as surrogate markers for the confirmation of MS diagnosis. A combination of antibodies, lymphocyte activation or cytokine production with abnormal MRI may significantly increase the sensitivity and specificity of MS diagnosis.     

Localization of insulin in mouse tissues using fluorescence microscopy and light microscope and high resolution autoradiography.

Summary Starved and fed mice were injected intravenously with either fluorescent or¹²⁵I-insulin and the localization of the hormone was investigated in various tissues. The fluorescent hormone was found in the kidney of starved animals only, whereas the radioactive hormone was found in the proximal convoluted tubule cells of the kidney of both fed and fasted animals, although the latter group appeared to contain more radioactivity than the first. With high resolution techniques the radioactivity in the kidney was found in the brush border, the apical vacuoles, the apical mitochondria and the nucleus, and never in the Golgi apparatus or the lysosomes. All other tissues contained radioactivity due to the hormone, but no difference in either the amount or the localization was found between fed an starved animals. It is concluded that the process of absorption of insulin by the cells of the proximal convoluted tubules is specific and that the hormone is not degraded in a way similar to other proteins, but is likely to be stored and to control cellular metabolic processes from its sites of localization.

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Lokalisierung von Insulin in Mäusegeweben durch Fluoreszenzmikroskopie sowie Licht- und Autoradiographie mit hohem Auflösungsvermögen

Zusammenfassung Gefütterte und fastende Mäuse erhielten fluoreszierendes oder mit¹²⁵J markiertes Insulin i.v. Die Lokalisation des Insulins in den Geweben wurde untersucht. Nur bei Tieren im Hungerzustand fand sich das fluoreszierende Hormon in den Nieren, während sich das radioaktive Insulin bei gefütterten und fastenden Tieren in den proximalen Tubulusschlingen der Nieren nachweisen ließ. Die fastenden Tiere schienen dabei mehr Radioaktivität aufzuweisen. Wurden die Nieren unter Verwendung von Techniken mit hohem Auflösungsvermögen untersucht, so fand sich die Radioaktivität im Bürstensaum, in den apikalen Vakuolen und Mitochondrien und dem Kern, jedoch nicht im Golgi-Apparat oder den Lysosomen. Alle anderen Gewebe enthielten Hormon-Radioaktivität, aber weder ihre Menge noch ihre Lokalisation unterschieden sich bei gefütterten und fastenden Tieren. Es wird gefolgert, daß der Prozeß der Insulinabsorption durch die Zellen der proximalen Tubulusschlingen spezifisch ist und daß das Hormon nicht in ähnlicher Weise wie andere Eiweißkörper abgebaut wird. Wahrscheinlich kommt es über eine Speicherung zu einer Steuerung der entsprechenden Stoffwechselprozesse in den subzellulären Organellen, in denen das Hormon sich befindet.

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Localisation de l'insuline dans les tissus de souris en utilisant la microscopie à fluorescence, le microscope optique et l'autohistoradiographie à haute résolution

Résumé La localisation d'insuline marquée avec I¹²⁵ ou avec de l'isothiocyanate de fluorescéine a été étudiée au moyen d'autohistoradiographie au microscope optique, au microscope électronique et au moyen de microscopie à fluorescence dans les tissus de souris à jeun et de souris alimentées normalement. L'hormone fluorescente a été trouvée dans les cellules des tubules proximaux du rein de souris à jeun seulement, tandis que l'hormone radioactive a été repérée aussi dans le rein de souris alimentées, bien qu'en quantité inférieure à celle trouvée chez les souris à jeun. Au microscope électronique la radioactivité est présente dans le rein dans la bordure en brosse, les vacuoles et les mitochondries apicales et le noyau, mais pas dans l'appareil de Golgi ou dans les lysosomes. —L'hormone radioactive est aussi présente dans les autres tissus mais sans différence quantitative entre les souris à jeun et les souris alimentées. Les auteurs concluent que le procédé de ré-absorption de l'insuline par les cellules des tubules proximaux du rein est spécifique: l'hormone n'est pas dégradée de la même façon que les autres protéines mais probablement stockée afin de contrôler les processus métaboliques relevant des organelles subcellulaires dans lesquelles elle se trouve.

     

发光二极管荧光显微镜实验室诊断效果评价

目的评估发光二极管荧光显微镜实验室诊断结核分枝杆菌的能力。方法收集409痰标本,以固体罗氏培养为金标准,比较发光二极管荧光显微镜与普通光学显微镜、普通荧光显微镜在敏感性、特异性及检出时间上的差异,并比较技术人员对几种方法的评价。结果罗氏培养阳性的175份痰标本中,普通光学显微镜法112份为阳性,普通荧光显微镜法121份为阳性,发光二极管荧光显微镜法130份为阳性,敏感性分别为64.0%,69.1%,74.3%;罗氏培养阴性的234份痰标本中,普通光学显微镜法233份为阴性,荧光显微镜法227份为阴性,发光二极管荧光显微镜法227份为阴性,特异性分别为99.6%,97.0%,97.0%。χ2检验统计结果表明,发光二极管荧光显微镜与普通光学显微镜、普通荧光显微镜检测结果差异有统计学意义(P0.001)。普通光学显微镜、普通荧光显微镜和发光二极管荧光显微镜读片所用的读阳性片时间分别为186.7 s、75.1 s、72.2 s,发光二极管荧光显微镜与普通光学显微镜在读阳性片时间上差异有统计学意义(P0.05),与普通荧光显微镜差异无统计学意义。参与研究的技术员评价发光二极管荧光显微镜较其他2种方法易接受。结论发光二极管荧光显微镜较普通光学显微镜及普通荧光显微镜在检查效果及检出时间上存在明显优势,对技术人员的亲和性好,具有推广使用的价值。     

Visualization of Endothelial Clefts and Nuclei in Living Microvessels with Combined Reflectance and Fluorescence Confocal Microscopy

Objective: The cellular basis for spatial heterogeneity along postcapillary venules in their response to inflammatory mediators is not understood. To study permeability regulatory processes on an individual cell basis in intact microvessels, we developed methods to delineate individual endothelial cells within living, single perfused microvessels. Methods: Individual postcapillary microvessels in the mesenteries of frogs and hamsters were perfused with chloride-free Ringer's solutions containing AgNO₃ (0.1 g per 100 ml) for 5–10 s. Vessels were immediately flushed with Ringer's solution containing serum albumin and some vessels subsequently perfused with the fluorescent nucleic acid stain YO-PR0-1 (1 μM) for 10–15 min. Vessels were imaged in situ using laser scanning confocal microscopy. A reflectance image from silver precipitate in the endothelial clefts and a fluorescence image of the nuclei were simultaneously recorded. Stacks of confocal images were merged and used to reconstruct the three-dimensional orientation of endothelial cells. Hydraulic conductivity of some frog mesenteric venular vessels, measured before and after perfusion with AgNO₃, was used to assess the integrity of the vessels. Results: The endothelial cell clefts were delineated with a very fine, readily imaged precipitate of silver. Hydraulic conductivity measured after AgNO₃ perfusion in frog mesenteric venular microvessels was not significantly different from control. Vessels showed a slight reduction in reactivity to inflammatory stimuli. The three-dimensional pattern of endothelial cells in the living vessels was imaged. Conclusions: Combining confocal microscopy with silver staining in single perfused microvessels was shown to be an effective means to delineate the three-dimensional pattern of endothelial cells forming microvessel walls. It enables further study of the vascular structure and function relationships at the individual cell level in intact microvessels.     

Proton magnetic resonance spectroscopy reveals similar white matter biochemical changes in patients with chronic hypertension and early Alzheimer's disease

OBJECTIVES: Hypertension is a risk factor for dementia and is associated with some of the brain changes that are found in Alzheimer's disease and other neurodegenerative diseases, such as atrophy and neurofibrillary tangles. We evaluated the cerebral white matter biochemical pattern in healthy older subjects, older patients with chronic hypertension, and patients with Alzheimer's disease (AD) using proton magnetic resonance spectroscopy (1H-MRS). DESIGN: Cross-sectional study. SETTING: University-affiliated outpatient clinic. PARTICIPANTS: Ten healthy older subjects, 10 cognitively intact older patients with chronic hypertension, and 10 older patients with early AD. MEASUREMENTS: All subjects underwent clinical examination, neuropsychological assessment, and 1H-MRS to measure N-acetylaspartate (NAA), myoinositol, choline, and creatine resonance signals in an 8-cm3 voxel located in the paratrigonal white matter region bilaterally. NAA/creatine, myoinositol/creatine, and choline/creatine ratios were measured, and the mean values were compared using one-way analysis of variance with Tukey test for post hoc analysis. RESULTS: A significantly higher mean myoinositol/creatine (ratio +/- standard deviation) was found in hypertensive patients (0.67 +/- 0.05) and in AD patients (0.68 +/- 0.08) than in controls (0.56 +/- 0.04) (P <.001). Conversely neither NAA/creatine ratio nor choline/creatine ratio differed among the three groups. CONCLUSIONS: In this study, cognitively intact chronic hypertensive older patients had a higher white matter myoinositol/creatine ratio compared with healthy older subjects, suggesting that myoinositol may be a sensitive marker of the effects of chronic hypertension on the brain. Moreover, the similar increase of myoinositol/creatine ratio in patients with hypertension and in those with early AD provides further evidence of common brain changes with these conditions.     

Fluorescence spectroscopy incorporated in an Optical Biopsy System for the detection of early neoplasia in Barrett's esophagus

Endoscopic surveillance is recommended for patients with Barrett's esophagus (BE) to detect high‐grade intraepithelial neoplasia (HGIN) or early cancer (EC). Early neoplasia is difficult to detect with white light endoscopy and random biopsies are associated with sampling error. Fluorescence spectroscopy has been studied to distinguish non‐dysplastic Barrett's epithelium (NDBE) from early neoplasia. The Optical Biopsy System (OBS) uses an optical fiber integrated in a regular biopsy forceps. This allows real‐time spectroscopy and ensures spot‐on correlation between the spectral signature and corresponding physical biopsy. The OBS may provide an easy‐to‐use endoscopic tool during BE surveillance. We aimed to develop a tissue‐differentiating algorithm and correlate the discriminating properties of the OBS with the constructed algorithm to the endoscopist's assessment of the Barrett's esophagus. In BE patients undergoing endoscopy, areas suspicious for neoplasia and endoscopically non‐suspicious areas were investigated with the OBS, followed by a correlating physical biopsy with the optical biopsy forceps. Spectra were correlated to histology and an algorithm was constructed to discriminate between HGIN/EC and NDBE using smoothed linear dicriminant analysis. The constructed classifier was internally cross‐validated and correlated to the endoscopist's assessment of the BE segment. A total of 47 patients were included (39 males, age 66 years): 35 BE patients were referred with early neoplasia and 12 patients with NDBE. A total of 245 areas were investigated with following histology: 43 HGIN/EC, 66 low‐grade intraepithelial neoplasia, 108 NDBE, 28 gastric or squamous mucosa. Areas with low‐grade intraepithelial neoplasia and gastric/squamous mucosa were excluded. The area under the receiver operating characteristic curve of the constructed classifier was 0.78. Sensitivity and specificity for the discrimination between NDBE and HGIN/EC of OBS alone were 81% and 58% respectively. When OBS was combined with the endoscopist's assesssment, sensitivity was 91% and specificity 50%. If this protocol would have guided the decision to obtain biopsies, half of the biopsies would have been avoided, yet 4/43 areas containing HGIN/EC (9%) would have been inadvertently classified as unsuspicious. In this study, the OBS was used to construct an algorithm to discriminate neoplastic from non‐neoplastic BE. Moreover, the feasibility of OBS with the constructed algorithm as an adjunctive tool to the endoscopist's assessment during endoscopic BE surveillance was demonstrated. These results should be validated in future studies. In addition, other probe‐based spectroscopy techniques may be integrated in this optical biopsy forceps system.     

POLArIS,a versatile probe for molecular orientation,revealed actin filaments associated with microtubule asters in early embryos

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArIS^act, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArIS^act with FPM. In living starfish early embryos expressing POLArIS^act, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner.

Many biomolecules function as molecular assemblies such as protein complexes, nucleic acids, and lipid bilayers. Their function often depends on the changes in molecular arrangements of their constituents, for example, conformational changes during movement of molecular motors along cytoskeletons (1–3), rotation of F₁-ATPases (4), and bending/extending of integrins (5, 6). Molecular orientation is the key information to study the relationship between such mutual changes of molecular arrangements and their function. Fluorescence microscopy has been widely used to study the structures and the dynamics of biomolecules in living cells, but monitoring the orientation of fluorescently labeled molecules in living cells is challenging even with the latest fluorescent microscopy including super-resolution approaches. Fluorophores, such as fluorescent proteins and organic compound dyes, emit fluorescence by radiating dipoles. The light emitted from a single dipole is fully polarized along the dipole axis. Therefore, when fluorescent labels are rigidly bound to molecules, the orientation of molecules can be monitored by analyzing the polarization state of the fluorescence. Fluorescence polarization microscopy (FPM) has been used for this approach (1–9) and is especially useful for detecting the presence of orderly assembled biomolecules and changes in their arrangements (10–12). In addition, FPM can report molecular orientations at a single-molecule level even in living cells when target molecules are labeled at proper density (3, 7). Super-resolution FPM methods have also been recently reported (13–16).Despite such promising advantages and advancements in methodology, FPM has not been widely used in biomedical research because of the difficulties in the rotationally constrained labeling of target molecules with fluorescent proteins. To monitor the molecular orientation, a target molecule and a fluorescent protein must be rigidly connected so that the orientation of the target and that of the dipole of the fluorophore are sterically fixed. For many cases, intensive screening of the linkage was required for each target molecule since there was no universal method for constrained tagging due to difficulties in the rational three-dimensional design of protein connection. A novel, more versatile and easier approach is therefore imperative for expanding the application of FPM.For this purpose, we have developed a versatile molecular orientation probe, named the Probe for Orientation and Localization Assessment, recognizing specific Intracellular Structures of interest (POLArIS). POLArIS is a high-affinity recombinant binder rigidly connected to a fluorescent protein. The recombinant binder can be screened by phage display to specifically target biomolecules of interests. Thus, POLArIS can tag biomolecules of interest with fluorescent proteins in a rotationally constrained manner. As a proof of principle, we developed a POLArIS that specifically binds to F-actin (POLArIS^act) and demonstrated that POLArIS^act reports the orientations of actin assembly in living cells. Moreover, by observing starfish embryos expressing POLArIS^act, we found an F-actin–based subcellular architecture that associates with the microtubule aster during mitosis, which had not been previously reported, demonstrating that POLArIS^act is useful for detecting ordered structures made of actin filaments in living cells.     

Genotyping of human platelet antigen-1 by gene amplification and labelling in one system and automated fluorescence correlation spectroscopy

Genotyping of human platelet antigen-1 (HPA-1) is required for the diagnosis and appropriate therapy of alloimmunization. Recently, the HPA-1 polymorphism has been identified as an inherited risk factor for thrombosis. Most currently used methods for HPA-1 genotyping have the disadvantage of time-consuming post-polymerase chain reaction (PCR) processes such as ligation (oligonucleotide ligation assay), restriction enzyme digestion (allele-specific restriction enzyme analysis) and electrophoresis (single-strand conformation polymorphism). We present a novel method for HPA-1 genotyping based on a homogeneous PCR strategy (GALIOS, gene amplification and labelling in one system) combined with automated fluorescence correlation spectroscopy (FCS). The PCR uses one pair of gene-specific amplification primers and two allele-specific, semi-nested labelling primers. The allele-specific labelling primers differ in a single nucleotide (T for HPA-1a/1a, C for HPA-1b/1b) and are coupled to different fluorescent dyes. The quantities of generated fluorescent PCR products are analysed by FCS at 543 nm and 633 nm excitation wavelength respectively. The genotypes determined using this method were in 100% concordance with the results obtained by allele-specific restriction analysis (n = 380 samples). The assay was validated for specificity, reliability and the dynamic range. This innovative method of rapid HPA-1 genotyping offers a specific and robust system, which is applicable for routine HPA-1 genotyping.     

Metabolic changes of anterior cingulate cortex in patients with hepatic cirrhosis: A magnetic resonance spectroscopy study

Aim: The anterior cingulate cortex (ACC) plays an important role in cognitive functions. The purpose of this study is to compare metabolite concentrations in the ACC of cirrhotic patients with normal controls, and to correlate metabolite changes with Child–Pugh class and with severity of hepatic encephalopathy (HE). Methods: Fifty‐two cirrhotic patients and 30 healthy volunteers were included in this study. All subjects performed the number connection test type A (NCT‐A) and digital symbol test (DST) before multiple resonance (MR) examinations. Single‐voxel proton MR spectroscopy (MRS) data in the ACC were acquired on a 1.5‐T scanner. The ratios of all metabolites to creatine and phosphocreatine (Cr) were obtained. Statistical analysis was performed to evaluate the difference between control and cirrhotic patients, with respect to metabolite ratios. The correlation between metabolite ratios and Child–Pugh scale, severity of HE, venous ammonia and neuropsychiatric test results was analyzed. Results: The ratios of choline (Cho)/Cr and myo‐inositol (mIns)/Cr were significantly lower, and the ratio of glutamine– glutamate (Glx)/Cr was significantly higher in cirrhotic patients than those in controls (P < 0.001). mIns/Cr correlated negatively with Child–Pugh scale (r = −0.496, P < 0.001) and HE degree (r = −0.313, P < 0.05). Venous ammonia had a significant correlation with Cho/Cr (r = −0.329, P < 0.05) and mIns/Cr (r = −0.347, P < 0.05). No statistical correlation between metabolite ratios and neuropsychological tests was found for cirrhotic patients, but mIns/Cr did have a statistical correlation with NCT‐A (r = −0.270, P < 0.05) and DST (r = 0.463, P < 0.001) when all subjects were included in the analysis. Conclusion: Significant metabolite changes were seen in the ACC in cirrhotic patients. Of the metabolites examined, the mIns/Cr level in the ACC was most closely associated with the severity of HE and hepatic functional reserve reflected by Child–Pugh scale.     

糖尿病肾病患者血清脂蛋白C反应蛋白载脂蛋白检测及其临床意义

目的 检测2型糖尿病患者血清中半胱氨酸蛋白酶抑制剂C(CysC)、脂蛋白(a)[LP(a)]、载脂蛋白A1 (ApoA1)、载脂蛋白B(ApoB)和超敏C反应蛋白(hs- CRP)水平,并探讨其临床意义.方法 96例2型糖尿病患者分为单纯糖尿病组50例、糖尿病肾病组46例,另选择50例健康体检者作为对照组,分别检测各组血清中的CysC、Lp(a)、ApoA1、ApoB以及hs-CRP水平.结果 2型糖尿病组与对照组比较,CysC[(0.95±0.16)mg/L对(0.46±0.17)mg/L,P＜0.01]、LP(a) [(214.8±182.3)mg/L对(113.1±76.2)mg/L,P＜0.01]、hs- CRP[(2.57±1.84)mg/L对(1.07±0.38)mg/L,P＜0.01]、ApoB[(1.12±0.18)g/L对(0.81±0.15)g/L,P＜0.01]水平均升高;而ApoA1水平降低[(1.02±0.17)g/L对(1.27±0.14)g/L,P＜0.01].糖尿病肾病组CysC(2.14±0.68)mg/L、Lp(a)(455.6±263.5)mg/L、hs- CRP(7.24±4.55)mg/L、ApoB(1.22±0.17)g/L,均高于单纯糖尿病组(均P＜0.01).2型糖尿病患者尿微量白蛋白与hs- CRP (r=0.478,P＜0.01)、CysC(r=0.55,P＜0.05)、Lp(a) (r=0.487,P＜0.05)、ApoB(r=0.505,P＜0.05)呈正相关,与ApoA1(r=-0.52,P＜0.05)呈负相关.结论 CysC、Lp(a)、hsCRP、载脂蛋白可作为诊断糖尿病早期肾损害的敏感指标,联合检测更具实用价值.