SUBSURFACE MICROSCOPIC VISUALIZATION OF BRAIN-TISSUE IN-VIVO - PRESENT, PROBLEMS AND PROSPECTS

The location of the brain within the cranium has prevented in vivo stu dies with microscopical resolution for a long time. Cranial window tec hniques provide microscopical access to the brain surface, but subsurf ace structures cannot be visualized with conventional microscopy. Conf ocal microscopy with its increased depth of penetration and optical se ctioning capabilities is ideally suited for the investigation of thick biological specimens, Due to the scanning process, however, temporal resolution is limited, a significant disadvantage in the in vivo setti ng. In this article we demonstrate that confocal laser scanning micros copy can be utilized to study brain cortex microvascular morphology, c apillary hemodynamics, leukocyte behaviour and intracellular ion conce ntrations in anesthetized rats through a closed cranial window. Three different confocal microscopes are compared: a Biorad MRC-600 with mul tiline Kr/Ar-Laser (488/568/647 nm), a Noran Odyssey acousto-optic sca nning microscope with multiline Ar-Laser (458/488//514/529 nm) and a B iorad Viewscan DVC-250 slit scanning microscope with Ar-Laser (488/514 nm). With all microscopes a Zeiss x 40 water immersion objective, n.a . 0.75 is used. A Laser-Doppler flowmeter continuously measures region al cerebral blood flow in the area of microscopical investigation. As fluorescent dyes we used: fluorescein sodium as blood plasma marker (g iven I.V.); rhodamine 6G to label leukocytes (given I.V.); and the AM- esters of BCECF (pH-sensitive), Fluo-3 and Calcium-Green (both calcium -sensitive) as intracellular ion-concentration markers (loaded via sup erfusion over the cranial window). With this setup, we are able to stu dy the flow dynamics in the capillary network of the cortex (erythrocy te flow velocities and flux rates), the behaviour of leukocytes in cap illaries and postcapillary veins (plugging of capillaries, adhesion to the endothelium, extravasation into the parenchyma), and intracellula r changes of [H+] and [Ca2+] under physiological and pathophysiologica l conditions (cerebral ischemia and meningitis). The comparison betwee n the conventional CLSM (Biorad 600) and the real time CLSMs revealed that the increase in temporal resolution afforded by the real time ins truments is offset by a reduction in spatial resolution and, most impo rtantly, in the signal to noise ratio, resulting in a lower depth of p enetration into the tissue and necessitating frame averaging.