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Biomedical subjects

A Liljeborg

Publications and source records attributed to A Liljeborg.

13 recordsLinked to original sources
PubMed2000

Characterization of probe binding and comparison of its influence on fluorescence lifetime of two pH-sensitive benzo[c]xanthene dyes using intensity-modulated multiple-wavelength scanning technique.

Quantitative pH imaging using the carboxy seminaphthofluorescein dyes SNAFL-1 and SNAFL-2 can be performed by measurement of intensity ratios or fluorescence lifetimes. However, there is a controversy as to whether the latter method has the practical advantage of a straightforward pH calibration in buffers compared to a cumbersome and time-consuming procedure in cells. In this study we have undertaken a systematic study of the potential factors influencing the fluorescence lifetime of the probes at different pH using confocal microscopy. In vitro results demonstrate that factors such as lipid and protein concentrations have a substantial influence on pH measurements based on fluorescence lifetime. The pH could be overestimated by more than 2 pH units. Studies in permeabilized COS-7 cells demonstrate the same trends as observed in the in vitro studies.

Animals↗
PubMed2000

Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance.

We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL-2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8-bit digital image. However, the pixel-to-pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.

Animals↗
PubMed1999

Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging.

We have here sought to cross-correlate the expression of immunoreactivities for several neuropeptides, nitric oxide synthase (NOS) and the growth associated protein GAP-43 in subpopulations of dorsal root ganglion (DRG) neurons tagged by the selective markers isolectin B4 and the neurofilament antibody RT97, selective for, respectively, subpopulations of small and large DRG neurons. By use of double- and triple-labeling immunohistochemistry, non-manipulated and sciatic nerve transected young adult rats as well as aged (30-months-old) rats were examined using a confocal microscope equipped with enhanced spectral separation. In young adult rats, the DRG neuron profiles could be divided into three subpopulations (B4 binding (B4+) approximately 50%; RT97-immunoreactive (RT97+) approximately 35%; B4-/RT97- approximately 15%). Calcitonin gene-related peptide (CGRP) is expressed in all three subpopulations. Galanin message-associated peptide (GMAP) colocalize with CGRP (100%) but is not expressed in RT97+ profiles. NOS is present in the RT97- subpopulations and frequently colocalize with CGRP (92%). GAP-43 is expressed in all three DRG subpopulations and colocalize with CGRP (88%), GMAP (38%) and/or NOS (22%). Only very small differences were seen among the young adult rats, implicating that the size of respective subpopulation as well as the expression pattern for neuropeptides, NOS and GAP-43 are fairly stable. Sciatic nerve transection reduced B4-binding but not RT97-like immunoreactivity. Distinct changes in the expression of neuropeptides, NOS and GAP-43 were evident in the DRG subpopulations and, furthermore, the regulatory changes were very similar among the lesioned animals. The relative size of the DRG subpopulations was unaffected by aging, while the expression of neuropeptides was altered showing similarities with the changes induced by axotomy in young adult rats.

Aging↗
PubMed1998

Interferon-gamma receptors are expressed at synapses in the rat superficial dorsal horn and lateral spinal nucleus.

Interferon-gamma can facilitate the spinal nociceptive flexor reflex and may elicit neuropathic pain-related behavior in rats and mice. Immunoreactivity for the interferon-gamma receptor (IFN-gamma R) occurs in the superficial layers of the dorsal horn and the lateral spinal nucleus in the rat and mouse spinal cord, as well as in subsets of neurons in the dorsal root ganglia. The aim of the present study was to examine the cellular localization and origin of the IFN-gamma R in the spinal cord. As viewed by confocal microscopy, the immunopositivity for the IFN-gamma R was co-localized with that of the presynaptic marker synaptophysin and with neuronal nitric oxide synthase in the lateral spinal nucleus, whereas only a minor overlap with these molecules was observed in laminae I and II of the dorsal horn. There was no co-localization of the IFN-gamma R with markers for astrocytes and microglial cells. Ultrastructurally, the IFN-gamma R was found predominantly in axon terminals in the lateral spinal nucleus, but at postsynaptic sites in dendrites in laminae I and II. The IFN-gamma R expressed in neurons in dorsal root ganglia was transported in axons both centrally and peripherally. Hemisection of the spinal cord caused no reduction in immunolabelling of the IFN-gamma R in the dorsal horn or the lateral spinal nucleus. Since rhizotomy does not affect the immunolabelling in the lateral spinal nucleus, our observation indicates that the presynaptic receptors in this nucleus are derived from intrinsic neurons. The localization of the IFN-gamma R in the spinal cord differed from that of the AMPA glutamate receptor subunits 2 and 3 and the substance P receptor (NK1). Our results, showing localization of IFN-gamma R to pre- and postsynaptic sites in the dorsal horn and lateral spinal nucleus indicate that IFN-gamma can modulate nociception at the spinal cord level.

Animals↗
PubMed1998

Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique.

We demonstrate the simultaneous recording of confocal lifetime images of multiple fluorophores. The confocal microscope used in the study combines intensity-modulated laser illumination, lock-in detection and spectral separation of the fluorescent light. A theoretical investigation is presented that describes how the signal-to-noise ratio (SNR) depends on various factors such as modulation frequency, degree of modulation and number of detected photons. Theory predicts that, compared with ordinary intensity images, lifetime images will have a SNR that is, at best, approximately four times lower. Experimental results are presented that confirm this prediction.

Journal Article↗
PubMed1996

Confocal 3-dimensional DNA image cytometry in thick tissue sections.

We present a three-dimensional confocal DNA image cytometry (3-D CICM) method for analysis of DNA content in 30-40-microns-thick sections of routinely processed paraffin-embedded specimens. A comparison of DNA ploidy profiles obtained by 3-D CICM and conventional DNA image cytometry (ICM) on tissue sections sections showed significantly higher numbers of cells with high DNA content in DNA histograms by 3-D CICM. As estimated by 3-D CICM, the size of nuclei frequently exceeded the thickness of tissue sections used in conventional ICM, which suggested that many nuclei measured by this technique may be incomplete. This artifact was excluded in 3-D CICM by automatic rejection of cut nuclear profiles. This and the favorable ratio of tissue thickness to nuclear size in 3-D CICM permitted the DNA quantitation even in large cells with highly increased DNA ploidy values such as megakaryocytes and Reed-Sternberg cells of Hodgkin's disease. Additionally, 3D-CICM allowed evaluation or morphometric parameters and 3-dimensional reconstruction of studied cells.

Bone Marrow↗
PubMed1995

A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope.

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.

Animals↗
PubMed1989

Native fibrin gel networks observed by 3D microscopy, permeation and turbidity.

Native fully hydrated fibrin gels formed at different fibrinogen and thrombin concentrations and at different ionic strengths were studied by confocal laser 3D microscopy, liquid permeation and turbidity. The gels were found to be composed of straight rod-like fiber elements that often came together at denser nodes. In gels formed at high fibrinogen concentrations, or with high amounts of thrombin, the spaces between the fibers decreased, indicating a decrease of gel porosity. The fiber strands were also shorter. Gel porosity decreased dramatically in gels formed at the high ionic strengths. Shorter fibers were observed and fiber swelling occurred at ionic strengths above 0.24. Quantitative parameters for gel porosity, fiber mass/length ratio and diameter were also derived by liquid permeation and turbidometric analyses of the gels. Permeation analysis showed that gel porosity (measured as Ks) decreased in gels formed at higher fibrin and thrombin concentrations in agreement with the porosity observed by microscopy. The turbidometric analysis showed good agreement with the permeation data for gels formed at various thrombin concentrations, but supported the permeation data more poorly in gels formed at different fibrinogen concentrations, especially above 2.5 mg/ml. Turbidometric analysis showed that the fiber mass/length ratio and diameter decreased in gels formed at ionic strength up to 0.24, as was seen in the permeation study. However, at higher ionic strengths swelling of the fibers was suggested from the gel turbidity data and this was also indicated by microscopy. These findings are discussed in relation to previous hydrodynamic and electron microscopic studies of fibrin gels.

Fibrinogen↗
PubMed1989

A confocal laser microscope scanner for digital recording of optical serial sections.

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illuminatin, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinated scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024x1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (about 1 microm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.

Animals↗
PubMed1988

Three-dimensional reconstruction of neurons in the lamprey spinal cord in whole-mount, using a confocal laser scanning microscope.

When investigating the detailed morphology of nerve cells, three-dimensional structural information is often of great value. We present here a technique by which 'optical sectioning' and three-dimensional reconstruction of fluorescence-labelled neurons in the lamprey spinal cord has been performed by means of a confocal microscope scanner with a laser beam as the light source. In confocal microscopy only a small spot of the specimen is being illuminated at any one time, and only light from the illuminated spot is detected. This gives several advantages compared with traditional microscopy: (1) Lateral resolution is improved, and any 'halo' effects occurring around structures with intense fluorescence are drastically reduced. In addition (2), a unique depth resolution is obtained due to the strong attenuation of structures that are out of focus. This allows the system presented here to (3) perform a detailed three-dimensional computer reconstruction of the neuron, without any need for physical sectioning of the tissue. The volume of data points sampled can subsequently be treated in various ways, including selection of different viewing angles, enhancement of contours, and background suppression.

Animals↗
PubMed1985

Autoradiographic changes of 3H-alpha-bungarotoxin binding in rat hind limb muscles after a cryoinjury of the sciatic nerve and in acrylamide intoxication.

The morphological distribution of acetylcholine receptors in rat tibialis anterior and soleus muscles after a cryoinjury of the sciatic nerve was studied by in vitro autoradiography with 3H-alpha-bungarotoxin (3H-alpha-Btx). Tibialis anterior from rats intoxicated with acrylamide was also investigated by this technique. A computer system was used for densitometry and colour coding of the autoradiographs. In normal muscle, 3H-alpha-Btx binding was observed only at the motor endplates. From the second day after the nerve lesion the area of binding spread from the motor endplate region towards the muscle-tendon regions and on the 16th day almost all parts of the muscles showed increased binding. The binding had begun to decrease in the middle parts of the muscles on the 20th day and on the 30th day binding was observed only in the motor endplate area. Tibialis anterior from rats intoxicated with acrylamide (total dose 1100 mg X kg-1) showed 3H-alpha-Btx binding over the entire muscle.

Acrylamide↗
PubMed1985

The laser reflection method. Computerized analysis of speckle pattern.

The laser reflection method of remote measurements permits registration of small tooth movements. Its precision was investigated by studying the errors associated with patient repositioning in the apparatus using the computer controlled image scanner OSIRIS. Fifty repositionings of healthy subjects were evaluated. The results show that the mean error of repositioning is 2.8 micron laterally and 9.2 micron sagittally. This agrees with earlier manual-visual measurements but the higher precision due to computer evaluation of the speckle patterns gives much more reliable estimate of the repositioning error. The study confirms the fact that the laser reflection method permits accurate measurements of tooth movements.

Adult↗