Technical Approaches

In-situ Confocal Microscopy.

Our laboratory is equipped with a number of scanning disk confocal microscopes which allow us to image real time in depth vascular tissue and interacting white blood cells. In the image above arteriolar wall is visualized by positioning the focal plan in the middle of the vessel (top). By positioning the focal plane on the upper part of the vessel the endothelial cells (EC) lining can be seen (bottom). The vessel was stained EC surface receptor ICAM-1 to outline the ECs.

In-situ Immunofluorescent Labeling.

In a cremaster muscle venule, we labeled the EC surface receptor PECAM-1 (green) to outline the endothelial junctions and CD11A (beta2-integrin, which is expressed on all white cells, red) to visualize circulating leukocytes. This approach allows us to quantitatively study leukocyte-endothelial cells interactions. ICAM-1 expression patterns in control and TNF-alpha treated mouse cremaster muscle arterioles was quantified using in-situ immunofluorescent labeling.  The image above demonstrates the expression patterns (expression relatively proportionate to the surface expression) of one of the important adhesion molecules on the EC surface during control or inflammatory conditions.

Permeability of blood vessels.

To quantify the permeability coefficients of the vascular wall in live mice, blood vessels were perfused with fluorescently labels albumin solution during a 40 minute time period.  The extravasation of albumin molecules from the lumen and their accumulation in the surrounding tissue is demonstrated in the movie below.

Ca2+ imaging.

A common way to study cell-cell communication is to quantify intracellular calcium behavior. To do so in live animals, microvessels were loaded with Fluor-4 molecules which bind all free intracellular Ca2+. In situ blood vessels were imaged confocally in anesthetized mice.  In the following movie, Ca2+ release from the intracellular stores into the cytosol can be seen following topical stimulation with acetylcholine.

Quantitative Hematology.

To identify and quantify subpopulations of white blood cells, a number of techniques can be utilized. In the image above, a smear preparation were made and stained to differentiate between neutrophils (left) and lymphocytes (right). Additionally, leukocyte subpopulations can be immuno-labeled in-situ with specific surface markers and later identified in a blood sample based on their fluorescence.

Flow Dynamics: In-situ/Simulation.

To assess the flow profile in unbranched venules, fluorescently labeled beads (0.5 mm) were injected intravenously into the blood stream. Bead trajectories were visualized as shown above. The outlines are placed at the approximate location of firmly adhered leukocyte; the figure shows that the bead trajectories are excluded from a near-wall region.

Individual Myocyte Contraction Coupling.

When muscle contracts, signals are sent to the adjacent microvessel which can then respond by dilating.