Hardware and methodology for targeting single brain arterioles for photothrombotic stroke on an upright microscope

Investigators have begun to probe the role of individual surface arterioles in maintaining both the structure and function of cortical regions using vessel-specific clotting by in vivo photothrombosis after craniotomy in mice. To induce targeted strokes we describe a simple adaptation of a commercial upright Olympus BX51WI microscope, permitting light from a 532 nm laser to be directed into the back aperture of a high numerical aperture fluorescence objective. The system involves using a filter slot available on an Olympus BX series microscope to direct a collimated laser beam through the normal epifluorescence path to the objective back aperture resulting in focused photoactivation, with lateral and axial dimensions less than 3 and 5 μm, respectively. Existing fluorescence filters and dichroic mirrors are employed permitting one to safely target the green laser beam and view the clotting process based as red epifluorescence, either through the eye pieces or using a CCD camera. Interruption in blood flow can be confirmed using laser speckle microscopy. The positioning of the photothrombotic laser in this manner does not impede subsequent analysis of brain microcirculation using two-photon microscopy or other imaging methods. It is conceivable that this modification and laser system can also be used for other scenarios where targeted photoactivation or photobleaching would be required.