Dual-beam laser illuminator of fluorescence microscope for in vivo microcirculation studies

A new fluorescence intravital microscope of long working distance (39 mm) has been developed for the observation of microcirculation in a wide visual field by designing a simple epi-illumination technique with dual laser beams. Cross-illumination, in which a pair of laser beams is symmetrically placed on either side of the objective such that they intersect at the focal plane of the objective, was employed to produce uniform distribution of the incident light in the object plane. In vitro experiments using a fluorescein isothiocyanate dextran (FITC-dextran; molecular weight = 70000) solution of known concentration confirmed uniform tracer excitation in a wide visual field (approximately 30 mm²), and a linear correlation between fluorescence intensity and tracer concentration (r = 0.999), ranging between 5 μmol l^-1 and 25 μmol l^-1. In vivo observations in the microcirculation of a hamster cheek pouch indicated that the present technique had the advantage of high contrast compared with the image obtained by bright-field transillumination. This microscope illuminator may prove useful for the evaluation of vascular permeability under physiological and inflammatory conditions, with sufficient quantitative reliability to determine tracer concentrations in all parts of the microvascular network. Furthermore, a long working distance in this technique could have considerable advantages for the application to nail-fold capillaroscopy in humans.