MEASUREMENT OF ABSOLUTE TRACER CONCENTRATIONS IN TISSUE-SECTIONS BY USING DIGITAL IMAGING FLUORESCENCE MICROSCOPY - APPLICATION TO THE STUDY OF PLASMA-PROTEIN UPTAKE BY THE ARTERIAL-WALL

Digital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wa ll. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be deri ved from digitized images. These investigations may be relevant to oth er studies employing DIFM. Nonlinearities were found to arise from off sets in the video digitizers, from background fluorescence and stray l ight within the microscope and from the transfer characteristics of th e intensified CCD camera. Camera gain controls showed complex behaviou r. Camera output fell substantially for several hours after switching on and was affected by room temperature. Large spatial variations in r esponse were caused by the geometry of the microscope optics and by th e image intensifier. However, the ratios between areas were not affect ed by light intensity or camera gain settings. Measured intensities we re-independent of the depthwise location of fluorophores within tissue sections but they were affected by the emission from objects outside the measuring area. Photobleaching of tracer varied significantly over the range of excitation intensities and durations used but was not co ncentration dependent. Methods used to correct these effects and obtai n a uniform, linear and constant relationship between concentration an d grey level are described. Using the system and appropriate correctio ns, in vivo uptake of sulphorhodamine-B-labelled serum albumin by the rabbit aortic wall was investigated. Results obtained for the mean upt ake of tracer and its local variation were quantitatively similar to t hose previously obtained with nonmicroscopic methods.