The development of multi-photon intravital microscopy, in particular two-photon microscopy (2PM), has been a breakthrough technique for deep-tissue imaging of dynamic cell behavior inside live organisms and has substantially advanced the field of immunology. However, intravital time-lapse imaging over prolonged time periods is complicated by slow tissue drifts caused by vital activity, leading to shifting fields of views and making the acquired image sequence partially or completely unanalyzable. To solve this issue, we have established a system that performs continuous drift offset correction in real time using fine pattern matching during 2PM acquisition. We incorporated an extensive use of graphical processing unit (CPU) for high-speed computing required for real time correction during data acquisition. This allowed us to perform prolonged acquisitions and increase the proportion of analyzable datasets to nearly 100% in lymphoid and non -lymphoid tissues. Considering the straightforward implementation of our newly developed system, we anticipate that it will be applicable for other users interested in improving the quality of live imaging data acquisition. (C) 2016 Elsevier B.V. All rights reserved.