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Biomedical subjects

K Carlsson

Publications and source records attributed to K Carlsson.

At least 19 recordsLinked to original sources
PubMed1995

Improved fluorophore separation with IMS confocal microscopy.

Some of the most extensively used fluorophores for multicolour fluorescence labelling of tissue, such as Lucifer yellow, fluorescein, Cyanine-3.18 and rhodamine, all have the disadvantage of being difficult to separate spectrally due to their broad and overlapping emission spectra. With the intensity-modulated multiple-beam scanning (IMS) technique, cross-talk between labels can be reduced to a fraction of that seen using conventional excitation and detection technique. Thus, cross-talk to the 'red' channel decreased with about one order of magnitude for the 'green' fluorophores Lucifer yellow and FITC. For the tested 'red' fluorophores, the amount of cross-talk to the green channel was reduced by 60-90%.

Animals↗
PubMed1995

Fibrinogenemia Tampere--a dysfibrinogenemia with defective gelation and thromboembolic disease.

Fibrinogen Tampere was found in a woman with severe thromboembolic disease. The thrombin induced clotting time of her plasma and purified fibrinogen was slightly prolonged. The activation of fibrinogen Tampere appeared to be normal but subsequent gelation was defective. We studied fibrin gels formed at different ionic strengths and at different fibrinogen and calcium concentrations by liquid permeation, turbidity, and 3D laser microscopy. Crosslinking was studied by SDS-gel electrophoresis. The gels formed from fibrinogen Tampere were at ionic strength above 0.2 much tighter and had lower fiber mass-length ratios than normal gels as judged by permeability and turbidity data. At ionic strength 0.15 and at different calcium concentrations analysis by permeability showed the same results for fibrinogen Tampere as for normal gels. Analysis by turbidity at ionic strength 0.15 suggested swelling of the fibers at low calcium concentrations. 3D microscopy revealed perturbed clot architecture under all conditions. In fibrin gels from fibrinogen Tampere, the gamma-chain crosslinking was normal but the crosslinking of alpha-chains was delayed at ionic strength 0.2 and also at lower ionic strengths on lowering the calcium concentration. The abnormal gelation may be due to a mutation in the fibrinogen molecule. Tendency to form tight fibrin gels and/or insufficient crosslinked fibrin matrix may be pathogenetic in this thrombotic disease.

Adult↗
PubMed1995

A confocal scanning laser microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusions in living cells stained with Fura-2.

A confocal scanning laser microscope (CSLM) for observation and quantitative ratiometric measurements of the intracellular dynamics of Ca2+ ions in living neurons has been developed. The instrument consists of a UV-enhanced CSLM, an optical arrangement providing simultaneous excitation at two wavelengths, an electronic arrangement for processing the simultaneous fluorescence response, and software for computing the absolute Ca2+ concentrations, ([Ca2+]). The instrument can be used for any excitation ratiometric measurements, provided that the dye substance used is excitable by wavelengths between 334 nm and 750 nm (such as, e.g. Fura-2). The spatial resolution of the CSLM, as well as a temporal resolution of 20 ms per line (maximum sampling rate) for dynamic measurements are provided by the instrument. Using Fura-2 in calibrated Ca2+ buffer solutions, the instrument measures [Ca2+] between 0 and 1.35 mumol.l-1 with an error of less than 1%. The capability of the instrument to measure absolute [Ca2+] was verified by recording fluorescence images of test solutions with well defined [Ca2+] values (Molecular Probes, Eugene, Ore., USA, C-3009 calibration solutions). In order to verify the dynamic capability of the instrument in real biological specimens, fluorescence changes of Fura-2 that were due to an intracellular flux of Ca2+ ions, and to an increase of [Ca2+]i (the intracellular Ca2+ concentration) have been recorded in Fura-2-loaded cultured cells of the line TE 671.

Calcium↗
PubMed1995

Pathologic fibrin formation and cold-induced clotting of membrane oxygenators during cardiopulmonary bypass.

In 1,800 patients undergoing cardiac surgery over a 2-year period, 11 incidents of abnormal inlet pressure elevations occurred before the membrane oxygenators. In 3 patients, the oxygenators had to be changed during cardiopulmonary bypass. This complication was found to be caused by fibrin formation possibly secondary to precipitation of fibrinogen with other coagulation factors in the heat exchangers of the oxygenators during the cooling phase. Large amounts of fibrin were demonstrated in the heat exchanger of the oxygenators. After careful washing of the apparatus, plasmin was added and fibrin was detected by measuring D-dimer levels. In heat exchangers from uneventful operations, only trace amounts of fibrin were found. Because there were no cold agglutinins demonstrated in the patients before surgery, cryoprecipitation studies were performed soon after surgery. When the patients' plasma samples were studied at different temperatures, from 37 degrees C down to 3 degrees C, cryoprecipitates or a gel (in 1 patient only) were formed. This indicated that there might be something abnormal with regard to fibrinogen-fibrin formation. The study patients were therefore investigated after the acute phase of the operation had ended for various coagulation factors, as well as for fibrin gel network characteristics. The results were compared with those of a control group (n = 10) with uneventful operations. There were no differences between the groups with regard to levels of coagulation factors VII and VIII and von Willebrand factor, although they were increased in both groups. The mean levels of coagulation inhibitors, antithrombin and Protein S, were slightly lower in the study patients. All of these patients had a highly pathologic, ie, tight fibrin gel network, except for the patient in whose sample a gel formed, despite being treated with aspirin or oral anticoagulants. The network was also tighter in some of the controls (v middle-aged reference individuals), although it was significantly tighter in the patients. It is concluded that some individuals who have an increased tendency to form tighter fibrin gel networks might be at increased risk for a severe complication during cardiac surgery performed under hypothermia.

Aged↗
PubMed1995

The affinity of troxerutin for the venous wall measured by laser scanning microscopy.

The uptake and localization of troxerutin, a trihydroxy-ethyl-rutoside, in the venous wall have been studied in patients undergoing long saphenous vein surgery. Troxerutin, an autofluorescent drug, is currently used to relieve oedema and subjective symptoms in patients with chronic venous insufficiency. In order to determine the localization of the troxerutin, a confocal scanning laser microscope has been used to record the fluorescence from vein cross sections. The quantified fluorescence was used as a measure of the local concentration of troxerutin. In order to reduce the effects of local variation, several images have been scanned from each specimen. Then the recorded data have been analysed to see how the fluorescence varies in the radial direction within the venous wall. Results showed that troxerutin was significantly accumulated in both inner and outer parts of the venous wall. Whereas inner wall troxerutin uptake resulted from direct diffusion through the lumen, the outer wall uptake proceeded likely from the vasa vasorum circulation.

Adult↗
PubMed1994

Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation.

The porosity, fiber dimension and architecture of fibrin gels formed in recalcified plasma on addition of thrombin are, within a certain range of thrombin concentrations, determined by the initial rate of fibrinogen activation. Furthermore, the initial network formed in this range creates the scaffold into which subsequently activated fibrinogen molecules are deposited. Change in thrombin concentration that occurs during gelation, as a result of indigenous thrombin generation in plasma, does not qualitatively alter this scaffold. The formation of the networks obeys a more complex rule when low amounts of thrombin are added or with recalcified plasma without added thrombin. These networks are tighter than would be expected from the initial rate of fibrinogen activation. In this case an extremely porous network is probably formed initially, followed by formation of a secondary, superimposed network of a less porous architectural quality. The latter structure appears to be governed by the rate of indigenous generation in plasma of thrombin-like enzymes in combination with the particular type of fibrinmonomers being produced. In addition our findings establish the rules for proper determination of gel structures in clinical plasma samples. The sequelae of a variety of clot structures that may be formed in vivo are discussed.

Blood Coagulation↗
PubMed1994

Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminolevulinic Acid↗
PubMed1994

Simultaneous confocal recording of multiple fluorescent labels with improved channel separation.

Confocal microscopes are often used to study specimens labelled with fluorophores. A commonly used method for simultaneous recording of the distribution of multiple fluorophores is to divide the fluorescent light emitted by the specimen into different wavelength regions using dichroic and bandpass filters. These different wavelength regions are then distributed to multiple detectors. However, the broad and overlapping spectra of commonly used fluorophores often result in considerable crosstalk between channels. A new technique, intensity-modulated multiple-beam scanning (IMS) microfluorometry, can be used to reduce this cross-talk substantially. The IMS technique is implemented with two laser beams of different wavelengths, intensity-modulated at different frequencies, which illuminate the specimen simultaneously. The two laser wavelengths predominantly excite one fluorophore each. Fluorescent light from the specimen is divided into two wavelength regions (red and green) which are detected by two photomultiplier tubes. The output signals from the photomultiplier tubes are connected to lock-in amplifiers. The effect of using modulated laser beams, in combination with lock-in amplifiers, is strongly to reduce cross-talk between the channels. The performance of the IMS technique using various types of specimen is compared with the results obtained using the conventional multi-detector method.

Animals↗
PubMed1993

Methods for large data volumes from confocal scanning laser microscopy of lung.

Confocal scanning laser microscopy makes it possible to obtain series of optical sections in precise registration. Certain studies of lung parenchyma, however, require both the fine resolution obtainable with high-numerical-aperture (NA) objectives and the extensive fields of view that usually would be achieved only with low-NA objectives. This article presents a technique that resolves this conflict by using a sequence of operations: (i) to correct intensity variations on individual sections due to non-uniform illumination/detection characteristics of the microscope; (ii) to correct intensity variations between successive sections in a series due to, for example, depth-related absorption or step changes in detector sensitivity; (iii) to adjust adjacent, overlapping stacks of sections to a common intensity level; and (iv) to fuse a group of such overlapping stacks into a single series of larger sections. This resulting stack may contain, for example, a complete cross-section of an alveolar ductal unit about 500 microns or more in diameter at about 1-micron pixel resolution.

Algorithms↗
PubMed1992

Uptake and localisation of O-(beta-hydroxyethyl)-rutosides in the venous wall, measured by laser scanning microscopy.

The uptake and localisation of O-(beta-hydroxyethyl)-rutosides (HR) in the venous wall was studied in 8 patients undergoing crossectomy for a varicose long saphenous vein. The fluorescence of cross-sections of the vein wall was measured by laser scanning microscopy, based on the autofluorescence of HR. Four patients (treated group) received 2 x 1.5 g HR IV before surgery, and four (untreated patients) served as controls. Uptake of HR into the veins from treated patients was seen, with a mean fluorescence intensity of 80.9 units compared to 49.4 units in the untreated veins. The increase in fluorescence was clearly demarcated on the endothelial side of the vein wall. It is concluded that HR passes into the vascular wall, where it is localised in the endothelial and sub-endothelial areas.

Adult↗
PubMed1992

Stimulation of NK cell, T cell, and monocyte functions by the novel immunomodulator Linomide after autologous bone marrow transplantation. A pilot study in patients with acute myeloid leukemia.

Immunostimulatory therapy is at present considered after autologous bone marrow transplantation (ABMT) in order to mimic the allogeneic graft-versus-leukemia effect and thereby reduce the relapse rate. In a pilot study, five adults with acute myeloid leukemia were treated with the new immunomodulator Linomide post-ABMT. Linomide (0.3 mg/kg/week orally) was given in cycles of three weeks followed by three weeks of rest for up to six months. During treatment periods cyclic increases of CD56+CD3- and CD16+ NK cells were observed in parallel with enhanced cytotoxic activity of patient cells against both the NK-sensitive K562 and NK-resistant Daudi cell lines. A cyclic increase of CD14+ monocytic cells was also recorded. The proliferative responses of patient cells to PHA and allogeneic cells (MLC) were enhanced during Linomide therapy. The in vitro production of TNF alpha, IFN gamma, and IL-1 followed the same cyclic increase during treatment periods. Side effects were generally mild, and no harmful effects on engraftment were seen. Linomide therapy after ABMT thus induces a broad immunostimulation that offers a potential benefit with regard to leukemia-free survival.

Adjuvants, Immunologic↗
PubMed1992

Quantitative analysis of Langerhans' cells in epidermis at irritant contact reactions using confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was used for quantitative analysis of CD1a+ cells in epidermis at irritant reactions. Sodium lauryl sulphate (2% and 4%) or non-anoic acid (20% and 80%) were applied to the skin of healthy volunteers under occlusion for 24 h. Skin biopsy specimens were taken after additional 24 h and were snap frozen. Freeze-sections, 25 microns thick, were stained with anti-CD1a antibodies (Leu-6) followed by FITC-labelled rabbit anti-mouse IgG. The sections were viewed and optically sectioned in the CLSM at four depth levels. The data was analysed using a threshold value for the fluorescence. The obtained result is presented as the proportion of specimen area having a fluorescence intensity above the threshold. The result demonstrates that the CLSM is a useful tool for obtaining not only structural information but also quantitative information from a defined tissue volume. In the present investigation it was possible to demonstrate variations in CD1a+ reactivity in epidermis at detergent-induced irritant reactions with a marked decrease in CD1a+ after 80% non-anoic acid exposure and only minor differences in the CD1a+ after 2% and 4% sodium lauryl sulphate exposure.

Cell Count↗
PubMed1991

Partial gastric corpectomy results in hypergastrinemia and development of gastric enterochromaffinlike-cell carcinoids in the rat.

Studies in the rat have shown that partial gastric corpectomy, in which about 75% of the acid-producing oxyntic mucosa was removed, leads to markedly reduced acid secretion and a feedback increase in the plasma gastrin levels. Ten weeks after operation, the gastric enterochromaffin (ECL)-like cell density in the remaining part of the oxyntic mucosa had increased significantly. In the present study, the effects on the gastric ECL cells of lifelong persistent hypergastrinemia induced by partial (75%) corpectomy have been investigated. Seventy-five partially corpectomized rats and 40 control rats were investigated for plasma gastrin and oxyntic mucosal changes in a 124-week study. The partially corpectomized rats showed increased plasma gastrin levels after the operation; the mean increase compared with the controls was almost 10-fold during the entire study. The remaining oxyntic mucosa of the partially corpectomized rats differed from that of control rats in two respects, showing first general hypertrophy and second a marked hyperplasia of argyrophil ECL cells. The degree and incidence of these changes increased towards the end of the study, i.e., in the aging rats. An age-related increase in ECL-cell density occurred spontaneously also in the control rats but to a lesser extent than in the partially corpectomized group. ECL-cell carcinoids were found in the oxyntic mucosa of 26 of the 75 partially corpectomized rats. The first carcinoid was found 78 weeks after the beginning of the study. Six rats with carcinoids (23%) were found before week 104 (2 years) and the remainder, 20 (77%), were discovered later. No carcinoid tumor was found in the control rats. It is concluded that lifelong hypergastrinemia induced by partial corpectomy leads to the development of ECL-cell carcinoids in the oxyntic mucosa of some rats towards the end of their life span. This observation strongly supports the hypothesis that the gastric ECL-cell carcinoids found in rats treated with antisecretory drugs are caused by long-standing hypergastrinemia developing secondary to inhibition of gastric acid secretion.

Animals↗
PubMed1991

Imaging of fluorescent neurons labelled with fluoro-gold and fluorescent axon terminals labelled with AMCA (7-amino-4-methylcoumarine-3-acetic acid) conjugated antiserum using a UV-laser confocal scanning microscope.

This paper describes the implementation of an ultraviolet (UV) laser (Spectra Physics 171-18 with 3 lines: 334, 351 and 364 nm in UV) as light source for fluorescence confocal scanning microscopy. With this instrument it is possible to use fluorophores not previously available for confocal laser microscopical imaging of fluorophores such as fluoro-gold and AMCA. In the study we show confocal laser microscopical imaging of fluorescent motoneurons labelled by retrograde transport of fluoro-gold and AMCA-fluorescent axon terminals labelled with antisera against immunogenes as thyrotropin-releasing hormone (TRH) and calcitonin gene-related peptide (CGRP). These two fluorophores may be recorded simultaneously or separately by using a filter that suppresses the emission of one of the fluorophores. The described instrument should also be useful in applications involving detection of monoamines by the Falck-Hillarp technique, as well as measurements of cytosolic free calcium by indicators such as Fura-2 and Indo-1. Measurements performed in reflected and fluorescence light indicated that the resolution along the optical axis improved by about 25% when UV (351 nm) is used instead of visible light (514 nm). This figure is close to that expected on theoretical basis. There are, however, also serious problems related to the use of UV excitation. Firstly, objectives must be selected based on their UV transmission properties. Secondly, chromatic aberration may cause a substantial focal shift between illuminating and emitted light, calling for a flexible instrumental design in order to allow for compensation. As shown here, this problem can be circumvented by using reflecting objectives but at a price of lower resolution compared with high-aperture refracting objectives.

Animals↗
PubMed1991

Three-dimensional specimen reconstruction by confocal microscopy and digital image processing.

In a confocal microscope point-wise illumination of the specimen is used, often in the form of a focused laser beam that is scanned in a raster pattern. The reflected or fluorescent light from the specimen is focused onto a small aperture in front of the detector. This imaging method has advantages such as improved resolution and less stray light. In addition optical sectioning is possible, whereby thick specimens can be studied layer by layer without the need for mechanical sectioning. By combining information from a large number of optical sections, registered at different depths, a three-dimensional reconstruction of the specimen can be made. Digital image processing can be used both for producing images for visual display and for extracting quantitative information. Quantitative evaluation is hampered in many cases by factors such as absorption, scattering, and refraction of the light travelling through the specimen volume.

Image Processing, Computer-Assisted↗
PubMed1990

Native fibrin gel networks and factors influencing their formation in health and disease.

Hydrated fibrin gels were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels from normal fibrinogen were found to be composed of straight rod-like fiber elements which sometimes originated from denser nodes. In gels formed at increasing thrombin or fibrinogen concentrations, the gel networks became tighter and the porosity decreased. The fiber strands also became shorter. Gel porosity of the network decreased dramatically in gels formed at increasing ionic strengths. Shortening of the fibers were observed and fiber swelling occurred at ionic strength above 0.24. Albumin and dextran, when present in the gel forming system, affected the formation of more porous structures with strands of larger mass-length ratio and fiber thickness. This type of gels were also formed in plasma. Albumin and lipoproteins may be among the determinants for the formation of this type of gel structure in plasma. Gels formed when factor XIIIa instead of thrombin was used as catalyst for gelation showed a completely different structure in which lumps of polymeric material were held together by a network of fine fiber strands. Our studies have also shown that the methodologies employed may be useful in studies of gel structures in certain dysfibrinogenemias as well as in other diseases. We give examples of two patients with abnormal fibrinogen and of patients with ischaemic heart disease.

Adult↗