The development of confocal laser scanning methods for the study of vascular structure, function and receptor distribution

The work contained within this thesis describes the development of confocal laser scanning methods as applied to the study of blood vessel structure and receptor distribution. Prior to the start of the project there were no suitable techniques available for the study of 3D structure and spatial distribution of receptors which therefore necessitated the development of novel methods. The first phase of the work employed fluorescent DNA stains which provided an increased contrast and an ability to identify all cell types, number, orientation and viability within a living vascular wall. This nuclear staining method was then extended by introducing confocal analysis of myograph mounted arterial segments. The confocal method was successful in detecting areas of apparently disorganised smooth muscle cells within the wall of SHR basilar arteries. In addition, CLSM detected changes in the media of human arteries taken from cases of critical limb ischaemia and in the adventitia of mouse tail artery where the aib-adrenoceptor had been deleted. Structural studies of the vascular wall required the use of image analysis methods for quantification and feature extraction. This required the development of novel methods specific for, CLSM-derived, 3D volumes of vascular structure. A semi-automated thresholding and segmentation algorithm (IMTS) was developed and tested. The imaging and segmentation phase of the research aided in the identification of problems associated with quantification of 3D volumetric structures. Fluorescent-ligands can be used to identify high affinity binding sites within cells. A significant proportion of this thesis describes the development of fluorescence- binding using CLSM and image analysis. The results document the developmental work leading up to the construction of the first 'specific' binding curve to be performed on a single living cell. This paves the way for ligand-binding-type experiments on cells dissociated from biopsies or other small tissue samples. Overall, the project has established the use of confocal microscopy for the study of blood vessel structure, function and receptor distribution. This thesis describes the key steps in the development of the techniques and hopefully serves as a guide to those interested in using confocal based methods for the study of blood vessels