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PubMed2006

Daucus carota L. mediated bioreduction of prochiral ketones.

Stereoselective reductions of ketones to secondary alcohols are of the utmost importance in organic synthesis. Very high selectivities are observed with traditional reducing agents, mainly based on boron or transition metals, complexed with chiral ligands. Bioreductions mediated by intact cells from cut plants, vegetables and fruits are attractive alternatives and could facilitate transition towards a more biobased economy. This emerging area highlights the recent results obtained in the aqueous bioreduction of prochiral ketones using carrot roots. The applications of this methodology to asymmetric protonation, dynamic kinetic resolution and the synthesis of biologically relevant targets are presented.

Alcohols↗

PubMed2000

True optical resolution beyond the Rayleigh limit achieved by standing wave illumination.

During the last decade, various efforts have been undertaken to enhance the resolution of optical microscopes, mostly because of their importance in biological sciences. Herein, we describe a method to increase the resolution of fluorescence microscopy by illuminating the specimen with a mesh-like interference pattern of a laser source and electronic postprocessing of the images. We achieve 100-nm optical resolution, an improvement by a factor of more than 2 compared with standard fluorescence microscopy and of 1.5 compared with confocal scanning.

Animals↗

PubMed1985

Magnets, molecules and medicine.

This article provides an introduction to high-resolution NMR with discussion of some of the important hardware considerations related to applying high-resolution NMR in medicine and biology. Experience has shown that multidisciplinary groups usually have to be formed to run NMR spectrometers dedicated for biological use, to cope with the demands imposed by the living systems under investigation and use of the spectrometer system. NMR imaging has obviously had a tremendous impact in medicine and many NMR imagers are being installed in hospitals throughout the world. In comparison to the already well established imaging techniques using CT, radioisotopes and ultrasound the overall experience gained so far is limited and there is undoubtedly a lot of work to be carried out before the usefulness of NMR imaging is fully assessed. The usefulness of NMR spectroscopy as a tool in in vivo biological research is clearly established. Originally based on 31P NMR studies the scope and extent of this type of investigation have now been enhanced by the use of 1H, 13C and 19F NMR and therefore will continue to extend our knowledge of metabolism, especially if NMR tracer studies with 13C and 19F can be shown to have real utility in an analogous fashion to radioisotopes. All of the nuclei discussed can be used to study the metabolism of various types of disease including carcinogenesis. With regard to medicine, the results obtained so far are certainly interesting and undoubtedly contributions will be made to the understanding of disease and related metabolism. At this stage, however, it is perhaps too early to say that NMR spectroscopy will become a routine tool in medicine but the rate of progress over the last ten years does suggest that the use of in vivo spectroscopy in medicine does have a significant future.

Adipose Tissue↗

PubMedDate not supplied

Biocompatible inkjet printing technique for designed seeding of individual living cells.

Inkjet printers are capable of printing at high resolution by ejecting extremely small ink drops. Established printing technology will be able to seed living cells, at micrometer resolution, in arrangements similar to biological tissues. We describe the use of a biocompatible inkjet head and our investigation of the feasibility of microseeding with living cells. Living cells are easily damaged by heat; therefore, we used an electrostatically driven inkjet system that was able to eject ink without generating significant heat. Bovine vascular endothelial cells were prepared and suspended in culture medium, and the cell suspension was used as "ink" and ejected onto culture disks. Microscopic observation showed that the endothelial cells were situated in the ejected dots in the medium, and that the number of cells in each dot was dependent on the concentration of the cell suspension and ejection frequency chosen. After the ejected cells were incubated for a few hours, they adhered to the culture disks. Using our non-heat-generating, electrostatically driven inkjet system, living cells were safely ejected onto culture disks. This microseeding technique with living cells has the potential to advance the field of tissue engineering.

Animals↗

PubMedDate not supplied

Computer-aided analyses of thalamocortical afferent ingrowth.

Segregation of thalamocortical afferent (TCA) fibers precedes barrel formation in rodent somatosensory cortex (Killackey and Leshin, 1975; Jeanmonod et al., 1981; Jensen and Killackey, 1987b; Senft, 1989; Erzurumlu and Jhaveri, 1990). Hypotheses about the arborization strategies followed by these ingrowing fibers have been generated from evaluation of labeled terminal fragments of mouse TCAs (Senft and Woolsey, 1991a). Those TCAs were of necessity truncated by the histological processing needed to observe them at high resolution. This fragmentation, along with biological variability, forces conclusions about single intact axons to be derived from populations of parts of arbors. To evaluate the hypotheses critically, we designed a computer program to quantify morphological aspects of labeled TCAs drawn with a camera lucida. We constructed algorithms to abstract, from fiber populations, properties minimally affected by truncation. Our program analyzes, and displays as histograms, fiber and branch densities and orientations. To represent these features by additional graphical means, "average" ingrowing TCAs were generated, based on the accumulated statistics of the traced fiber fragments. Quantitative descriptions of TCA populations from postnatal day 1 (PND1) through PND7 are presented. These analyses show that fibers and their branches accumulate with age within the cortical plate (emergent layer IV), and to a lesser extent within developing layer VI. Simultaneously, the distributions of these afferents within cortical laminae transform from uniform to patchy in the plane of the cortex. Peaks exhibit the periodicity typical of mature barrels. Branches become more numerous focally as and where layer IV barrels emerge. Individually traced arbors show reduced total widths consistent with progressive pruning of branches extending into territories of inappropriate barrels, both in layer IV and deeper in the cortex.

Animals↗

PubMed1994

Two-photon molecular excitation provides intrinsic 3-dimensional resolution for laser-based microscopy and microphotochemistry.

With the development of sensitive and specific fluorescent indicators, modern laser scanning microscopies enable visualization and measurement of submicron, dynamic processes inside living cells and tissues. Here we describe the working principles of new, nonlinear laser microscopies based on two-photon molecular excitation. In these techniques, a pulsed laser produces peak photon densities high enough that when focused into an appropriate medium, excitation by photon energy combinations can occur. For example, two red photons interacting simultaneously with a fluorescent molecule can excite within it a UV electronic transition, one corresponding to twice the energy of each single photon. Because the amount of two-photon excitation depends on the square of the local illumination intensity, this process exhibits a unique localization to the diffraction-limited spot of the beam focus. Elsewhere along the beam, excitation of background and photodamage is virtually nonexistent. Focal point localization of two-photon excitation lends to all visualization, measurement, and photopharmacology studies an intrinsic, three-dimensional resolution. We describe some preliminary biological applications, specifically, imaging of vital DNA stains in developing cells and embryos, imaging of cellular metabolic activity from NADH autofluorescence, spatially resolved measurements of cytoplasmic calcium ion activity, and optically induced micropharmacology using caged bioeffector molecules.

Animals↗

PubMedDate not supplied

FDG PET and PET/CT imaging in lymphoma and melanoma.

FDG PET imaging is rapidly gaining acceptance in numerous clinical applications in oncology. PET/CT is a new imaging tool, which takes advantage of the excellent anatomic resolution of CT and the biologic characterization provided by FDG PET which together results in improvement in accuracy of detecting the extent of disease, provide a guide to biopsy or surgery, allow early response evaluation and prognostication. This article will provide specific clinical applications for the utilization of these imaging modalities in both lymphoma and melanoma.

Fluorodeoxyglucose F18↗

PubMed2005

An X-ray microscopy perspective on the effect of glutaraldehyde fixation on cells.

X-ray microscopy (XRM) is the only microscopy technique that can provide high-resolution (30 nm) imaging of biological specimens without the need to fix, stain or section them. We aim to determine the effect, if any, of glutaraldehyde fixation on algae cells from the XRM perspective and thus provide beneficial information for both X-ray and electron microscopists on artefacts induced by glutaraldehyde fixation. Three species of microalgae, Microcystis aeruginosa, Anabaena spiroides and Chlorella vulgaris, were used in this study. XRM images were obtained from unfixed and glutaraldehyde-fixed cells and cell diameter and percentage X-ray absorbency were measured. The mean diameter of cells from fixed preparations was smaller than from unfixed preparations; the mean diameter of M. aeruginosa cells was significantly reduced from 3.92 microm in unfixed cells to 3.43 microm in fixed cells (P < 0.05); in C. vulgaris the diameter of cells was also significantly reduced from 3.50 microm in unfixed to 2.98 microm in fixed samples (P < 0.05); whereas there was no significant reduction in the diameter of A. spiroides cells (4.04-3.90 microm). The protein crosslinking mechanism of glutaraldehyde probably generated free water molecules, which play an important role in radiation damage induced by X-rays. This was seen as mass loss and cell shrinkage, which in the present study occurred more frequently in fixed cells than in unfixed cells. In addition, we demonstrated that the uptake of glutaraldehyde by cells makes all protein constituents in the cell organize into a closely packed configuration, thus causing a rise in the percentage of X-ray absorbency. In fixed cells, this rise was approximately by a factor of two compared with unfixed samples in which protein constituents inside the cell are arranged in their native form.

Anabaena↗

PubMed2003

Development and use of fluorescent protein markers in living cells.

The ability to visualize, track, and quantify molecules and events in living cells with high spatial and temporal resolution is essential for understanding biological systems. Only recently has it become feasible to carry out these tasks due to the advent of fluorescent protein technology. Here, we trace the development of highly visible and minimally perturbing fluorescent proteins that, together with updated fluorescent imaging techniques, are providing unprecedented insights into the movement of proteins and their interactions with cellular components in living cells.

Cell Physiological Phenomena↗

PubMed2006

Nuclear medicine procedures for the diagnosis of acute and chronic renal failure.

The focus of this review is on the current role of nuclear imaging studies in the clinical evaluation of patients with acute and chronic renal failure. In this setting nuclear imaging has two roles: diagnostic and prognostic, indicating that these methods are an essential component in the evaluation of renal diseases. The functional assessment of the kidney by nuclear medicine procedures is based on the use of radioisotopes bound to non-metabolized molecules with known pharmacokinetics. Renal scintigraphy is usually applied for the assessment of renal function expressed as glomerular filtration rate, effective renal plasma flow or more generally kidney perfusion. Newer methods rely on positron emission tomography, which allows the generation of images with higher resolution and absolute quantitation of biological processes such as transport activities, enzyme activities or angiotensin receptors.

Acute Kidney Injury↗

PubMed2002

Use of a blood substitute to determine instantaneous murine right ventricular thickening with optical coherence tomography.

BACKGROUND: A satisfactory imaging technique to determine regional wall thickening of the murine myocardium is not available. Although cardiovascular imaging with light offers a novel solution, application is problematic because scattering by erythrocytes causes significant optical attenuation. METHODS AND RESULTS: Optical coherence tomography (OCT) is a technique for detailed resolution imaging of highly scattering biological tissues. To reduce the high level of blood scattering, a method was devised whereby murine blood was replaced with a hemoglobin-based blood substitute. The scattering and absorption properties of in vitro preparations of whole blood and dilutions of blood with a blood substitute were determined with a spectrophotometer and an inverse-adding doubling algorithm. OCT imaging of the same dilutions demonstrated a significant reduction in scattering at a hematocrit <5%. A fiber-optic OCT imaging system was used to image the murine right midventricular free wall before and after isovolumic replacement with blood substitute. Strong light attenuation prevented full thickness imaging before replacement, whereas visualization of the full ventricular thickness was possible after replacement. Baseline and imaging hematocrits were 52.4+/-3.8% and 3.7+/-1.2%, respectively. End-systolic and end-diastolic thickness values were 0.458+/-0.051 mm and 0.352+/-0.047 mm. Percent thickening fraction was 30.8+/- 7.5%. CONCLUSION: Optical imaging of the intact beating murine right ventricle was substantially improved by isovolumic blood replacement with a hemoglobin-based blood substitute. Although the current study has been directed toward imaging the murine heart, a blood substitute may be applied to various optical diagnostic and therapeutic techniques under investigation in cardiovascular medicine.

Animals↗

PubMed1989

HLA gene amplification and hybridization analysis of polymorphism. HLA matching for bone marrow transplantation of a patient with HLA-deficient severe combined immunodeficiency syndrome.

The treatment of choice for certain immunodeficiency syndromes and hematological disorders is bone marrow transplantation (BMT). The success of BMT is influenced by the degree of HLA compatibility between recipient and donor. However, aberrant expression of HLA sometimes makes it difficult, if not impossible, to determine the patient's HLA type by standard serological and cellular techniques. We describe here the application of new molecular biological techniques to perform high resolution HLA typing independent of HLA expression. A patient with HLA-deficient severe combined deficiency was HLA typed using in vitro amplification of the HLA genes and sequence-specific oligonucleotide probe hybridization (SSOPH). Two major advances provided by this technology are:detection of HLA polymorphism at the level of single amino acid differences; and elimination of a requirement for HLA expression. Although the patient's lymphocytes lacked class II HLA proteins, polymorphism associated with DR7,w53;DQw2;DRw11a (a split of DR5), w52b (a split of DRw52);DQw7 were identified. The patient's class I expression was partially defective, and typing was accomplished by a combination of serological (HLA-A and -C) and SSOPH analysis (HLA-B). Complete patient haplotypes were predicted after typing of family members [A2;B35(w6); Cw4; DRw11a(w52b);DQw7 and A2;B13(w4); Cw6;DR7(w53); DQw2]. Potential unrelated donors were typed and a donor was selected for BMT.

Alleles↗

PubMed1989

Cytochemical demonstration of increased phospholipid content in cell membranes in chlorphentermine-induced phospholipidosis.

We recently introduced a novel cytochemical approach to high-resolution cytochemistry of phospholipids in biological tissues. The technique consists of adsorption of bee venom phospholipase A2 to colloidal gold particles (PLA2-gold complex) and subsequent application of this complex for localization of the enzyme substrate, i.e., glycerophospholipids. In the present study, this technique was applied at the post-embedding level, in both light (LM) and transmission electron microscopy (TEM), to investigate drug-induced phospholipidosis, an experimental disorder in which the lysosomal catabolism of phospholipids is inhibited. Rats received one week of daily treatment (40 mg IP/kg) with chlorphentermine (CP), a cationic amphiphilic drug known to induce phospholipidosis in several tissues. Glutaraldehyde- and osmium-fixed lung and kidney tissues from both treated and control animals, were embedded in Epon and sections processed for labeling by PLA2-gold. In CP-treated specimens the presence of large osmiophilic inclusions in several cell types of lung parenchyma and kidney cortex confirmed the onset of phospholipidosis. These inclusions were densely labeled by PLA2-gold at both LM and TEM levels. Two general types of abnormal inclusions were distinguished on the basis of their ultrastructure and labeling pattern by PLA2-gold, suggesting different content or configuration of phospholipids. Moreover, quantitative evaluation of labeling density over various membrane compartments in lung alveolar cells evidenced significantly increased phospholipid content after CP treatment. In type II pneumocytes, such increases were measured in membranes of the RER, Golgi complex, outer and inner nuclear envelope, and the basolateral and apical domains of the plasma membrane. In capillary endothelial cells, the basal and luminal domains of the plasma membrane also showed an increase in labeling density. These results further demonstrate the potential usefulness of the PLA2-gold technique for in situ ultrastructural localization of phospholipids in normal and pathological tissues.

Animals↗

PubMed1992

Experimental test of an analytical model of aberration in an oil-immersion objective lens used in three-dimensional light microscopy.

Oil-immersion microscope objective lenses have been designed and optimized for the study of thin, two-dimensional object sections that are mounted immediately below the coverslip in a medium that is index matched to the immersion oil. It has been demonstrated both experimentally and through geometrical- and physical-optics theory that, when the microscope is not used with the correct coverslip or immersion oil, when the detector is not located at the optimal plane in image space, or when the object does not satisfy specific conditions, aberration will degrade both the contrast and the resolution of the image. In biology the most severe aberration is introduced when an oil-immersion objective lens is used to study thick specimens, such as living cells and tissues, whose refractive indices are significantly different from that of the immersion oil. We present a model of the three-dimensional imaging properties of a fluorescence light microscope subject to such aberration and compare the imaging properties predicted by the model with those measured experimentally. The model can be used to understand and compensate for aberration introduced to a microscope system under nondesign optical conditions so that both confocal laser scanning microscopy and optical serial sectioning microscopy can be optimized.

Mathematics↗

PubMed2003

Dual-axes confocal microscopy with post-objective scanning and low-coherence heterodyne detection.

We present a dual-axes confocal microscope that employs postobjective scanning and low-coherence heterodyne detection to collect vertical cross-sectional images from biological tissue with high axial resolution, reduced noise from scattered light, deep tissue penetration, and a large dynamic range. This architecture can be scaled down to millimeter dimensions with microelectromechanical systems technology for performance of in vivo optical biopsy.

Biopsy↗

PubMed2004

Information bounds and optimal analysis of dynamic single molecule measurements.

Time-resolved single molecule fluorescence measurements may be used to probe the conformational dynamics of biological macromolecules. The best time resolution in such techniques will only be achieved by measuring the arrival times of individual photons at the detector. A general approach to the estimation of molecular parameters based on individual photon arrival times is presented. The amount of information present in a data set is quantified by the Fisher information, thereby providing a guide to deriving the basic equations relating measurement uncertainties and time resolution. Based on these information-theoretical considerations, a data analysis algorithm is presented that details the optimal analysis of single-molecule data. This method natively accounts and corrects for background photons and cross talk, and can scale to an arbitrary number of channels. By construction, and with corroboration from computer simulations, we show that this algorithm reaches the theoretical limit, extracting the maximal information out of the data. The bias inherent in the algorithm is considered and its implications for experimental design are discussed. The ideas underlying this approach are general and are expected to be applicable to any information-limited measurement.

Algorithms↗

PubMed2003

Distribution of deciduous stands in villages located in coniferous forest landscapes in Sweden.

Termination of fire along with active removal of deciduous trees in favor of conifers together with anthropogenic transformation of productive forest into agricultural land, have transformed northern European coniferous forests and reduced their deciduous component. Locally, however, in the villages, deciduous trees and stands were maintained, and have more recently regenerated on abandoned agricultural land. We hypothesize that the present distribution of the deciduous component is related to the village in-field/out-field zonation in different regions, which emerges from physical conditions and recent economic development expressed as land-use change. We analyzed the spatial distribution of deciduous stands in in-field and out-field zones of villages in 6 boreal/hemiboreal Swedish regions (Norrbotten, Angermanland, Jämtland, Dalarna, Bergslagen, Småland). In each region 6 individual quadrates 5 x 5 km centered on village areas were selected. We found significant regional differences in the deciduous component (DEC) in different village zones. At the scale of villages Angermanland had the highest mean proportion of DEC (17%) and Jämtland the lowest (2%). However, the amounts of the DEC varied systematically in in-field and out-field zones. DEC was highest in the in-field in the south (Småland), but generally low further north. By contrast, the amount of DEC in the out-field was highest in the north. The relative amount of DEC in the forest edge peaked in landscapes with the strongest decline in active agriculture (Angermanland, Dalarna, Bergslagen). Because former and present local villages are vital for biodiversity linked to the deciduous component, our results indicate a need for integrated management of deciduous forest within entire landscapes. This study shows that simplified satellite data are useful for estimating the spatial distribution of deciduous trees and stands at the landscape scale. However, for detailed studies better thematic resolution is needed to determine biologically relevant differences in quality of deciduous stands.

Environmental Monitoring↗

PubMed1988

Penetration and uptake of colloidal gold-labeled concanavalin A in the junctional epithelium of the rat.

A complex of colloidal gold and concanavalin A (CG-Con A) with various biological properties and high ultrastructural resolution was applied into the sulcus of rat molar gingiva and traced with an electron microscope for three hours to examine the cytological changes occurring in the cells of the junctional epithelium (JE) during penetration of extrinsic irritants, and to determine the roles of JE cells in such a circumstance. While the penetration of CG-Con A was impeded on the surface of keratinized oral gingival/sulcular epithelium, CG-Con A penetrated swiftly through JE into the connective tissue. In the process of penetration, CG-Con A was taken up by lysosomal and vacuolar structures of JE cells in which degenerative changes were often provoked. Degeneration of JE cells was seen selectively in the second and/or third cell layers from the innermost cell layer of JE. It was assumed that JE cells by their phagocytic activity might participate in the first line of defense against extrinsic irritants. On the other hand, the phagocytic activity of JE cells seems also to be involved in tissue destruction, if the amount and/or toxicity of irritants exceed the dissimilating capacity of JE cells.

Animals↗