Two-photon microscopy with double-circle trajectories for in vivo cerebral blood flow measurements

Scanning microscopes normally use trajectories which produce full-frame images of an object at a low frame rate. Time-resolved measurements are possible if scans along a single line are repeated at a high rate. In conjunction with fluorescence labeling techniques, in vivo recording of blood flow in single capillaries is possible. The present work investigates scanning with double-circle trajectories to measure blood flow simultaneously in several vessels of a capillary network. With the trajectory centered near a bifurcation, a double circle crosses each vessel twice, creating a sensing gate for passing dark red blood cells in fluorescently labeled plasma. From the stack of scans repeated at 1,300 Hz, the time-resolved velocity is retrieved using an image correlation approach. Single bifurcation events can be identified from a few fluorescently labeled red blood cells. The applicability of the method for in vivo measurements is illustrated on the basis of two-photon laser scanning microscopy of the cerebral capillary network of mice. Its performance is assessed with synthetic data generated from a two-phase model for the perfusion in a capillary network. The calculation of velocities is found to be sufficiently robust for a wide range of conditions. The achievable limits depend significantly on the experimental conditions and are estimated to be in the 1 μm/s (velocity) and 0.1 s (time resolution) ranges, respectively. Some manual fine-tuning is required for optimal performance in terms of accuracy and time resolution. Further work may lead to improved reliability with which bifurcation events are identified in the algorithm and to include red blood cell flux and hematocrit measurements. With the capability for time-resolved measurements in all vessels of a bifurcation, double-circle scanning trajectories allow a detailed study of the dynamics in vascular networks.