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Kevin Braeckmans

Publications and source records attributed to Kevin Braeckmans.

9 recordsLinked to original sources
PubMed2007

Line FRAP with the confocal laser scanning microscope for diffusion measurements in small regions of 3-D samples.

We present a truly quantitative fluorescence recovery after photobleaching (FRAP) model for use with the confocal laser scanning microscope based on the photobleaching of a long line segment. The line FRAP method is developed to complement the disk FRAP method reported before. Although being more subject to the influence of noise, the line FRAP model has the advantage of a smaller bleach region, thus allowing for faster and more localized measurements of the diffusion coefficient and mobile fraction. The line FRAP model is also very well suited to examine directly the influence of the bleaching power on the effective bleaching resolution. We present the outline of the mathematical derivation, leading to a final analytical expression to calculate the fluorescence recovery. We examine the influence of the confocal aperture and the bleaching power on the measured diffusion coefficient to find the optimal experimental conditions for the line FRAP method. This will be done for R-phycoerythrin and FITC-dextrans of various molecular weights. The ability of the line FRAP method to measure correctly absolute diffusion coefficients in three-dimensional samples will be evaluated as well. Finally we show the application of the method to the simultaneous measurement of free green fluorescent protein diffusion in the cytoplasm and nucleus of living A549 cells.

Algorithms↗
PubMed2006

Characterization of diffusion of macromolecules in konjac glucomannan solutions and gels by fluorescence recovery after photobleaching technique.

Konjac glucomannan (KGM) is a neutral polysaccharide with interesting properties as gelling agent and thickener. Its peculiar biodegradability, being not degradable in the small intestine but degradable by the anaerobic human intestinal bacteria, turn it into a promising candidate for colonic drug delivery systems. In this study aqueous systems (0.5%, w/v,) of KGM from three different origins and their mixtures with xanthan gum (XG) (1:1) were evaluated as regards their rheological properties and the diffusion coefficients and mobile fraction of macromolecules (dextrans of different molecular weight). Rheological data illustrate the synergism between KGM and XG at a stoichiometric relationship 1:1. Moreover, fluorescence recovery after photobleaching (FRAP) data indicate that diffusion of probes through the polysaccharide systems cannot be completely explained by the macroscopic properties of the medium but it is related to their molecular size and as a consequence to a sieving mechanism. The strong differences between KGM from different suppliers suggest the convenience of establishing specifications for this material in order to use it as pharmaceutical excipient.

Dextrans↗
PubMed2005

Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching.

In this paper, the release of proteins from a novel self-gelling hydrogel based on biodegradable dextran microspheres is investigated. The protein-loaded macroscopic gels are obtained by hydration of mixtures of oppositely charged hydroxyethyl methacrylate-derivatized dextran microspheres with a protein solution. In media of low ionic strength (100 mM Hepes pH 7.0) it was found that the release of the entrapped model proteins (lysozyme, BSA and IgG) was slower than in saline (150 mM NaCl, 100 mM Hepes pH 7.0). The reason behind this observation is that substantial adsorption of the proteins onto the microspheres' surface and/or absorption in the microspheres takes place. Confocal images showed that independent of their crosslink density the microspheres are impermeable for BSA and IgG. BSA, bearing a negative charge at neutral pH, was adsorbed onto the surface of positively charged microspheres. Lysozyme, which is positively charged at neutral pH, was able to penetrate into the negatively charged microspheres. In saline, the gels showed continuous release of the different proteins for 25 to 60 days. Importantly, lysozyme was quantitatively and with full preservation of its enzymatic activity released in about 25 days. This emphasizes the protein friendly technology to prepare the protein-loaded gels. Mathematical modeling revealed that protein release followed Fick's second law, indicating that the systems are primarily diffusion controlled. These results show that these hydrogels are very suitable as injectable matrix for diffusion-controlled delivery of pharmaceutically active proteins.

Adsorption↗
PubMed2005

Vitreous: a barrier to nonviral ocular gene therapy.

PURPOSE: Intravitreal injection of therapeutic DNA, complexed to nonviral carriers such as cationic liposomes, may be promising in the treatment of many severe retinal eye diseases. However, after intravitreal injection, such DNA/cationic liposome complexes-called lipoplexes (LPXs)-which are typically hundreds of nanometers in size, must first diffuse through the vitreous before they can reach the retina. The aim of this study was to elucidate whether vitreous is a barrier for the LPXs and to find strategies to overcome this barrier. METHODS: Fluorescent polystyrene nanospheres and LPXs were mixed with vitreous, and their mobility was monitored by fluorescence recovery after photobleaching (FRAP), a microscopy-based technique. The stability of LPXs and naked plasmid DNA in vitreous was studied by gel electrophoresis. RESULTS: We showed that polystyrene nanospheres, in our first experiments used as a model for the LPXs, do not diffuse freely into the vitreous but adhere to fibrillar structures in the vitreous, most likely to collagen fibers. Making the surfaces of the polystyrene nanospheres hydrophilic by attaching hydrophilic polyethylene glycol (PEG) chains at their surfaces circumvented the binding to fibrillar structures in the vitreous. FRAP revealed that "pegylated" polystyrene nanospheres, as long as they are smaller than 500 nm, are indeed mobile in the vitreous. It was further demonstrated that LPXs severely aggregate in vitreous and strongly bind to biopolymers in the vitreous, which immobilizes them completely. However, as observed for the polystyrene nanospheres, coating of the LPXs with PEG averted their aggregation in the vitreous and their binding to fibrillar structures. CONCLUSIONS: Modifying the surfaces of LPXs with hydrophilic PEG chains prevents them from aggregating in vitreous. In this way, LPXs are obtained that can freely move in vitreous, an absolute criterion for reaching the retina after intravitreal injection.

Animals↗
PubMed2003

Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope.

Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.

Algorithms↗
PubMed2003

Encoding microcarriers by spatial selective photobleaching.

Bead-based assays on very large numbers of molecules in gene expression studies, drug screening and clinical diagnostics, require the encoding of each of the microspheres according to the particular ligand bound to its surface. This allows mixing the uniquely encoded microspheres and subjecting them to an assay simultaneously. When a particular microsphere gives a positive reaction, the substance on its surface can be identified by reading the code. Previously reported techniques for colour encoding polymer microspheres only allow for a limited number of unique codes. Graphical encoding methods use metallic particles, which are rather uncommon in screening applications. Here, we demonstrate a new approach to encode polymer microspheres that are commonly used in screening applications, such as polystyrene microspheres, with a method that provides a virtually unlimited number of unique codes. Patterns can be written in fluorescently dyed microspheres by 'spatial selective photobleaching' and can be identified by confocal microscopy. Such encoded microparticles can find broad application in the collection and analysis of genetic information, high-throughput screening, medical diagnostics and combinatorial chemistry, and can also be used for labelling of consumer goods or as security labels to prevent counterfeiting.

Microspheres↗
PubMed2002

Encoding microcarriers: present and future technologies.

In answer to the ever-increasing need to carry out many assays simultaneously in drug screening and drug discovery, several microcarrier-based multiplex technologies have arisen in the past few years. The compounds to be screened are attached to the surface of microcarriers, which can be mixed together in a vessel that contains the target analyte. Each microcarrier has to be encoded to know which compound is attached to its surface. In this article, the methods that have been developed for the encoding of microcarriers are reviewed and discussed.

Biological Assay↗
PubMedDate not supplied

Anomalous photobleaching in fluorescence recovery after photobleaching measurements due to excitation saturation--a case study for fluorescein.

In this study we examine the implications of excitation saturation on fluorescence recovery after photobleaching (FRAP) experiments. In particular we present both experimental and theoretical evidence that fluorescein, one of the most frequently used fluorophores in FRAP, does not always comply with the basic assumptions that are made in many FRAP models: an invariant bleaching illumination intensity distribution (BID) in combination with first-order photobleaching kinetics. High light intensity levels, which are typical for the photobleaching phase of FRAP experiments, can cause excitation saturation of fluorescein in the excited triplet state. We show by experiments and computer simulations that under such saturating conditions the higher-order diffraction maxima of the BID substantially contribute to the photobleaching process and can no longer be neglected. As a result, the bleached regions are larger than expected theoretically from the FRAP models. Although this effect is not always directly evident from the FRAP experiments, neglecting it may shift the calculated diffusion coefficient by as much as over one order of magnitude. We present a discussion on the implications of this saturation effect on various types of FRAP models.

Algorithms↗