Patric Van Oostveldt

A fluorescence method for the determination of the molecular weight cut-off of alginate-polylysine microcapsules.

Abstract— Several applications of microcapsules for the encapsulation of living cells or macromolecules require well defined pore sizes. The molecular weight cut‐off of alginate‐polylysine microcapsules has been determined using a range of fluorescent labelled dextran molecules. The diffusion of the fluorescein isothiocyanate labelled (FITC)‐dextrans into the microcapsules was followed by fluorescence and confocal laser scanning microscopy. The permeability of microcapsules for FITC‐dextrans with a molecular weight of 4 700 daltons and the impermeability for FITC‐dextrans with a molecular weight of 40 500 daltons was confirmed with both techniques. Determination of the molecular weight cut‐off, using confocal laser scanning microscopy was more reliable and required a smaller sample than fluorescence measurements.

HETEROGENEOUS PHOTOBLEACHING IN CONFOCAL MICROSCOPY CAUSED BY DIFFERENCES IN REFRACTIVE INDEX AND EXCITATION MODE

The photobleaching of fluorescence emission during confocal laser scanning was studied on well-defined, stained objects [microspheres of polystyrene or fluorescent gels of fluorescein isothiocyanate (FITC)-labeled dextran] and on biological samples. X,Y laser scanning with confocal microscopy induces fundamental differences in exposure rate and time in different z-planes orthogonal to the optical axis. A heterogeneous bleaching rate was observed at different focal levels in the polystyrene spheres and in the gels. This phenomenon can be caused by refractive index differences or is correlated with a photobleaching rate, which is dependent not only on the excitation light intensity but also on the photon flux (total intensity per unit of time). Heterogeneous excitation induced by refractive index differences results in photobleaching differences but will not necessarily cause heterogeneous emission intensity. Altered emission originating from altered excitation will be annihilated if the emitted light returns to the image plane along the same inverse path, compensating for the proportional increase or decrease in excitation intensity with an increased or decreased emission intensity. High numerical aperture or increased scanning speed increases the photobleaching rate. This leads to the conclusion that photobleaching in confocal scanning laser microscopy is dependent on photon energy flux density (joule/m2s).

Digital titration: Automated image acquisition and analysis of load and growth of Chlamydophila psittaci

Traditionally, the amount of infective chlamydiae in a given sample is determined by inoculating dilution series into cell cultures and physically counting chlamydial inclusions. This approach is time consuming, tedious, and error prone, mainly when dealing with high titers. Therefore, this paper describes a largely automated technique that was developed to standardize the determination of chlamydial load in vitro. Cells are fixed at 36 h post‐inoculation and bacteria visualized using standard immunological detection methods. Consequently, for 81 microscopic fields, an image is recorded at the interpolated focal plane. These images are then automatically processed using an ImageJ plugin and the obtained results are imported into Excel to determine the number of inclusion forming units per mL in the sample. The main advantage of this technique is that no or minimal sample dilution is required, thus minimizing dilution errors. In addition, this technique was employed during the early, middle and late growth stages of the chlamydial developmental cycle and results correlated well (P < 0.01) with 16S rRNA values from previous experiments, thereby proving its suitability to follow chlamydial growth in vitro. The method described is highly suitable for high throughput titration of cell culture inoculated samples and assessment of possible antichlamydial effects of novel compounds throughout the chlamydial growth cycle. Microsc. Res. Tech., 2009.

Heterogeneous photobleaching in confocal microscopy

Photobleaching was studied during recording of confocal scanning laser microscopy. Studies on fluorescent gels of FITC-labeled dextran were used to evaluate differential bleaching along thez-axis. Differential bleaching along the z-axis was observed and it was seen that this was related to the numerical aperture of the objective in use. This points to the conclusion that photon energy flux density is an important parameter in photobleaching. To check if photon energy flux density heterogeneity is affected by local variation in the refractive index of the sample, photobleaching rates were calculated for different fluorescent objects (sections of seeds, animal cells stained with nuclear stains, immunocytochemistry preparations) and a pronounced similarity was found between photobleaching rates and DIC images.