4D in in vivo 2-photon laser scanning fluorescence microscopy with sample motion in 6 degrees of freedom.

2-Photon laser scanning microscopy (TPLSM) is often used for chronic in vivo studies. Small deviations in the sample orientation, however, make comparison of three-dimensional image stacks taken at different time-points challenging. When analysing changes of three-dimensional structures over time (4D imaging) this fundamental problem is one of the main limitations when complex structures are studied repetitively. We used an upright two-photon microscope complemented with a software-controlled stage-rotation instead of a conventional stage for chronic in vivo imaging in the brain of transgenic mouse models of Alzheimer's disease. Before every session an optimal imaging condition was successfully created by aligning the surface of the cranial window perfectly perpendicular to the laser beam. Deviations in the sample orientation between consecutive imaging sessions could be eliminated which improves conditions for chronic in vivo studies.