Image registration is often a required and a time-consuming step in blood flow analysis of large microscopic video sequences in vivo. In order to obtain stable images for blood flow analysis, frame-to-frame image matching as a preprocessing step is a solution to the problem of movement during image acquisition. In this paper, microscopic system analysis without fluorescent labelling is performed to provide precise and continuous quantitative data of blood flow rate in individual microvessels of nude mice. The performance properties of several matching metrics are evaluated through simulated image registrations. An automatic image registration programme based on Powell's optimisation search method with low calculation redundancy was implemented. The matching method by variance of ratio is computationally efficient and improves the registration robustness and accuracy in practical application of microcirculation registration. The presented registration method shows acceptable results in close requisition to analyse red blood cell velocities, confirming the scientific potential of the system in blood flow analysis.