Aims. Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM) which require longer acquisition times to obtain a quantitative readout. This can prevent functional FLIM-FRET imaging in deep organs. Methods. We present and benchmark a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. Results. We show that Galene is able to resolve intravital FLIM-FRET, hyperspectral and image correlation spectroscopy images of intra-abdominal organs in murine models and NADH autofluorescence imaging of human dermal tissue subject to a wide range of physiological motions. In particular, we use the tool to image cancer cells expressing a Src FRET biosensor in an intrasplenic model of pancreatic cancer metastasis through an optical window. We reveal the dynamics of Src activation during adhesion and observe a significant delay in Src activity after priming with Fasudil at early time points, providing a potential mechanism for the significant reduction in metastasis observed in earlier end point experiments. The extensive physiological motion observed in the liver means that it would be impractical to acquire this data without the correction provided by Galene. Conclusions. We provide a new tool to allow researchers to obtain insight into the earliest event in the metastatic cascade and as such a new avenue to insight into a critical event driving cancer colonisation for pancreatic cancer and more broadly other metastatic cancer types. We show that the software may be used in a broad range of intravital imaging experiments and imaging modalities and so enable functional imaging in situations where a stable imaging platform is not always possible.