Validation of urea as an endogenous reference compound for the in vivo calibration of microdialysis probes

Microdialysis is a widely used experimental technique, which offers the opportunity to measure drug and metabolite concentrations in the interstitial space fluid in animals and humans. However, microdialysis probes need to be calibrated in vivo to obtain a recovery factor, which describes the relative drug transfer across the membrane. Recently, novel time-saving calibration techniques, based on the use of reference compounds, have been developed. In particular, the use of endogenous urea levels has been advocated. In the present study we set out to validate the use of the urea reference technique for microdialysis probe calibration in healthy volunteers, employing glucose and paracetamol as model analytes. Urea calibration was compared with the results of two standard calibration techniques, i.e. the no net flux technique and the retrodialysis technique. For glucose, recovery values, calculated by the urea reference technique differed significantly from those values, which were assessed by the no net flux technique (p < 0.05), whereas for paracetamol recovery values did not differ significantly, albeit a high intramethod variability was observed (CV=66%). As a conclusion, we could not confirm the hypothesis that recovery values calculated by the urea reference technique provide equivalent results compared with standard calibration techniques.     

In situ dark field microscopy for on-line monitoring of yeast cultures

A new-type in situ probe has been developed to acquire dark field images of yeast in bioreactors. It has been derived from an in situ bright field microscope that is able to measure cell density in bioreactors during fermentation processes. The illumination part of the probe has been replaced with a dark field device, in which an aspheric condenser is used, so that high contrast dark field images can be obtained. The technique of second imaging is implemented to improve the sharpness of the images by means of a relay lens. This new in situ probe is expected to enable the evaluation of the cell viability without staining owing to modern image processing.     

Protein analysis with tetra-substituted sulfonated cobalt phthalocyanine by the technique of Rayleigh light scattering

This is the first report of cobalt-tetrasulfonatophthalocyanine (CoTSPc) as a probe of Rayleigh light scattering (RLS) to determine proteins at nanogram levels. A highly sensitive method has been developed for the determination of proteins by the light scattering technique on a common spectrofluorimeter, based on the fact that the weak RLS of CoTSPc can be greatly enhanced in the presence of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.10-34.3 microg x mL(-1) for both human serum albumin and bovine serum albumin, with detection limits of 15.5 and 13.9 ng x mL(-1), respectively. Moreover, there is almost no interference of any amino acids and metal ions. The method has been applied to the direct determination of total proteins in human serum samples, and the results were satisfactory with clinical data provided.     

Selective detection of superoxide anion radicals generated from macrophages by using a novel fluorescent probe

Quantitation of superoxide radical (O (2)(-).) production at the site of radical generation remains challenging. A simple method to detect nanomolar to micromolar levels of superoxide radical in aqueous solution has been developed and optimized. This method is based on the efficient trapping of O(2)(-). using a novel fluorescent probe (2-chloro-1,3-dibenzothiazolinecyclohexene), coupled with a spectra character-signaling increase event. A high-specificity and high-sensitivity fluorescent probe was synthesized in-house and used to image O(2)(-). in living cells. Better selectivity for O(2)(-). over competing cellular reactive oxygen species and some biological compounds illustrates the advantages of our method. Under optimal conditions, the linear calibration range for superoxide anion radicals was 5.03 x 10(-9)-3.33 x 10(-6) M. The detection limit was 1.68 x 10(-9) M. Fluorescence images of probe-stained macrophages stimulated with 4beta-phorbol 12-myristate 13-acetate were obtained successfully using a confocal laser scanning microscope.     

A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA)

Methods to detect the presence of coliform bacteria in drinking water usually involve a series of complex cultivating steps that are time-consuming and subject to external influences. For this reason, the new 16S rRNA probe has been developed in this study as an alternative detector PCR-ELISA technique that does not involve the culture of bacteria and that is able to detect, identify, and quantify the representative coliform species present in water samples. Our results indicate that this technique is both rapid (detection time of 4 h) and accurate (1.4% error rate). The limit of detection (LOD) was 5 CFU/100 ml for total coliforms, which meets the standards set by most countries for drinking water. Our comparative study demonstrated that this PCR-ELISA method is superior to current conventional methods in terms of detection time, LOD, and accuracy.     

Light and Life in Baltimore—and Beyond

Baltimore has been the home of numerous biophysical studies using light to probe cells. One such study, quantitative measurement of lateral diffusion of rhodopsin, set the standard for experiments in which recovery after photobleaching is used to measure lateral diffusion. Development of this method from specialized microscopes to commercial scanning confocal microscopes has led to widespread use of the technique to measure lateral diffusion of membrane proteins and lipids, and as well diffusion and binding interactions in cell organelles and cytoplasm. Perturbation of equilibrium distributions by photobleaching has also been developed into a robust method to image molecular proximity in terms of fluorescence resonance energy transfer between donor and acceptor fluorophores.     

Computer program for analyzing donor photobleaching FRET image series.

BACKGROUND: The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel-by-pixel fashion from the image series recorded on the donor-only and donor and acceptor double-labeled samples. Because of the large number of pixels and the time-consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements. METHODS: New evaluation software was developed to analyze pbFRET data for Windows platform in National Instrument LabVIEW 6.1. This development environment contains a mathematical virtual instrument package, in which the Levenberg-Marquardt routine is also included. As a reference experiment, FRET efficiency between the two chains (beta2-microglobulin and heavy chain) of major histocompatibility complex (MHC) class I glycoproteins and FRET between MHC I and MHC II molecules were determined in the plasma membrane of JY, human B lymphoma cells. RESULTS: The bleaching time constants calculated on pixel-by-pixel basis can be displayed as a color-coded map or as a histogram from raw image format. CONCLUSION: In this report we introduce a new version of pbFRET analysis and data processing software that is able to generate a full analysis pattern of donor photobleaching image series under various conditions. .     

Analysis of glycerophosphoinositol by liquid chromatography-electrospray ionisation tandem mass spectrometry using a beta-cyclodextrin-bonded column

Glycerophosphoinositol (GroPIns) has been demonstrated to have important roles in many intracellular regulatory processes. GroPIns has been analysed for many years by anion-exchange HPLC after radiolabelling of cells in culture, but no method has been developed, to our knowledge, for the direct detection and quantitation of the unlabelled compound in such biological samples. Here is reported a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the direct quantitative analysis of GroPIns that can indeed be applied to cell extracts. Analyses were performed on a beta-cyclodextrin-bonded HPLC column using a binary mobile phase of acetonitrile and 20 mM ammonium formate in water, which allowed direct on-line detection by tandem mass spectrometry in negative electrospray ionisation (ESI) mode. The method was applied to the quantitative analysis of GroPIns in selected rat cell lines after a two-phase acid extraction of cultured cells using external calibration. The potential matrix signal suppression effects were investigated by the parallel quantitation of GroPIns in extracts of selected cultured cell lines with both external calibration and the standard additions method. The accuracy data obtained demonstrated the feasibility of external calibration, so allowing a simpler and less time-consuming approach than that of the standard additions method.     

Automatic Region-Based Brain Classification of MRI-T1 Data

Image segmentation of medical images is a challenging problem with several still not totally solved issues, such as noise interference and image artifacts. Region-based and histogram-based segmentation methods have been widely used in image segmentation. Problems arise when we use these methods, such as the selection of a suitable threshold value for the histogram-based method and the over-segmentation followed by the time-consuming merge processing in the region-based algorithm. To provide an efficient approach that not only produce better results, but also maintain low computational complexity, a new region dividing based technique is developed for image segmentation, which combines the advantages of both regions-based and histogram-based methods. The proposed method is applied to the challenging applications: Gray matter (GM), White matter (WM) and cerebro-spinal fluid (CSF) segmentation in brain MR Images. The method is evaluated on both simulated and real data, and compared with other segmentation techniques. The obtained results have demonstrated its improved performance and robustness.     

Corrim-based alignment for improved speed in single-particle image processing

The technique of single-particle electron cryomicroscopy is currently making possible the 3D structure determination of large macromolecular complexes at constantly increasing levels of resolution. Work at resolution now attainable requires many thousands of individual images to be processed computationally. The most time-consuming step of the image-processing procedure is usually the iterative alignment of individual particle images against a set of reference images derived from a preliminary 3-D structure. We have developed an improved multireference alignment procedure based on interpolated cross-correlation images (corrims) that results in an approximately 8-fold acceleration of the iterative alignment steps. These corrims can be used to restrict the number of image-alignment calculations by narrowing down the set of reference images. Another improvement in alignment speed has been achieved by optimising the software and its implementation on many parallel processors. This new corrim-based refinement has been found to work well with two different alignment algorithms, the commonly used "fast alignment by separate translational/rotational searches" and "exhaustive alignment by polar coordinates."     

Pyrolytic Methylation-Gas Chromatography of Whole Bacterial Cells for Rapid Profiling of Cellular Fatty Acids

A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) profiles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the filament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for time-consuming subculturing) as well as analysis of FAME profiles directly from complex environmental samples.     

Direct radioimmunoassay of progesterone in plasma of farm animals.

A radioimmunoassay technique has been developed for measuring progesterone directly, without extraction, in 20-mul-aliquots of plasma. The assay was performed in disposable polystyrene test-tubes. Working range of the calibration curve was 0.3 to 10 ng/ml, the precision rangin from 30 to 6%, respectively. The results obtained by the presented method are fully comparable to those reported by others. This method has been applied to farm animals, but is also applicable to other species whose plasma has a low affinity for progesterone.     

Cone-beam breast computed tomography using ultra-fast image reconstruction with constrained,total-variation minimization for suppression of artifacts

Compressed sensing based iterative reconstruction algorithms for computed tomography such as adaptive steepest descent-projection on convex sets (ASD-POCS) are attractive due to their applicability in incomplete datasets such as sparse-view data and can reduce radiation dose to the patients while preserving image quality. Although IR algorithms reduce image noise compared to analytical Feldkamp-Davis-Kress (FDK) algorithm, they may generate artifacts, particularly along the periphery of the object. One popular solution is to use finer image-grid followed by down-sampling. This approach is computationally intensive but may be compensated by reducing the field of view. Our proposed solution is to replace the algebraic reconstruction technique within the original ASD-POCS by ordered subsets-simultaneous algebraic reconstruction technique (OS-SART) and with initialization using FDK image. We refer to this method as Fast, Iterative, TV-Regularized, Statistical reconstruction Technique (FIRST). In this study, we investigate FIRST for cone-beam dedicated breast CT with large image matrix. The signal-difference to noise ratio (SDNR), the difference of the mean value and the variance of adipose and fibroglandular tissues for both FDK and FIRST reconstructions were determined. With FDK serving as the reference, the root-mean-square error (RMSE), bias, and the full-width at half-maximum (FWHM) of microcalcifications in two orthogonal directions were also computed. Our results suggest that FIRST is competitive to the finer image-grid method with shorter reconstruction time. Images reconstructed using the FIRST do not exhibit artifacts and outperformed FDK in terms of image noise. This suggests the potential of this approach for radiation dose reduction in cone-beam breast CT.     

Gradient temperature hybridization using a thermocycler for RNase protection assays

The RNase protection assay (RPA) is a sensitive and quantitative assay for the detection of specific RNAs. Because some probes require an optimal temperature for efficient hybridization to the target RNAs, preliminary experiments to determine the optimal temperatures have to be done empirically, a process that is laborious and time-consuming. In order to simplify this process, a new gradient temperature hybridization procedure has been developed with the use of a thermocycler. This procedure eliminates the need for preliminary experiments to determine the optimal temperature of hybridization for a given probe.     

CT iterative reconstruction in image space: A phantom study

Although iterative reconstruction is widely applied in SPECT/PET, its introduction in clinical CT is quite recent, in the past the demand for extensive computer power and long image reconstruction times have stopped the diffusion of this technique. Recently Iterative Reconstruction in Image Space (IRIS) has been introduced on Siemens top CT scanners. This recon method works on image data area, reducing the time-consuming loops on raw data and noise removal is obtained in subsequent iterative steps with a smoothing process. We evaluated image noise, low contrast resolution, CT number linearity and accuracy, transverse and z-axis spatial resolution using some dedicated phantoms in single, dual source and cardiac mode. We reconstructed images with a traditional filtered back-projection algorithm and with IRIS. The iterative procedure preserves spatial resolution, CT number accuracy and linearity moreover decreases image noise. These preliminary results support the idea that dose reduction with preserved image quality is possible with IRIS, even if studies on patients are necessary to confirm these data.     

Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples

A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a “standard”) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples.     

Real-time imaging of nitric oxide production in living cells with 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacence by invert fluorescence microscope.

Although the importance of nitric oxide (NO) as a signalling molecule in many biological processes is becoming increasingly evident, many proposed and potential biological functions of NO still remain unclear. Bioimaging is a good technique to visualize observation of nitric oxide in biological samples. In this report, a fluorescent probe, 1,3,5,7-tetramethyl-2,6-dicarbethoxy-8-(3',4'-diaminophenyl)-difluoroboradiaza-s-indacence (TMDCDABODIPY), has been first applied to real-time image NO produced in PC12 cells, Sf9 cells and human vascular endothelial cells at the presence of l-arginine with inverted fluorescence microscope. NO production in the cells is successfully captured and imaged with fine temporal and spatial resolution. The results prove that the probe combined with inverted fluorescence microscope can be developed into a sensitive and selective method for further study of NO release from cells.     

Effectively desynchronizing deep brain stimulation based on a coordinated delayed feedback stimulation via several sites: a computational study

In detailed simulations we present a coordinated delayed feedback stimulation as a particularly robust and mild technique for desynchronization. We feed back the measured and band-pass filtered local filed potential via several or multiple sites with different delays, respectively. This yields a resounding desynchronization in a naturally demand-controlled way. Our novel approach is superior to previously developed techniques: It is robust against variations of system parameters, e.g., the mean firing rate. It does not require time-consuming calibration. It also prevents intermittent resynchronization typically caused by all methods employing repetitive administration of shocks. We suggest our novel technique to be used for deep brain stimulation in patients suffering from neurological diseases with pathological synchronization, such as Parkinsonian tremor, essential tremor or epilepsy.