Rapid overlapping-volume acquisition and reconstruction (ROVAR): automated 3D tiling for high-resolution, large...

Micrometer-scale three-dimensional data from fluorescence microscopes offer unique insight into cellular morphology and function by resolving subcellular locations of fluorescent dyes and proteins. To increase field-of-view size while using a high-resolution multiphoton microscope, we have created an automated system of rapidly acquiring overlapping image stacks from multiple fields-of-view along a nonplanar tissue surface. Each image stack is acquired only between the surface and the maximal penetrating depth, as determined by the image signal-to-background ratio. This results in the acquisition of the volume containing visible tissue along the tissue surface, excluding the empty volume above the tissue and the volume beyond the maximum imaging depth within the tissue. The automated collection of overlapping volumes is followed by reconstruction that can efficiently generate a single three-dimensional volume of the tissue surface. This approach yields data spanning multiple millimetres at micrometre resolution that is faster while requiring less work from the microscope operator. The advantages of the system are demonstrated by acquisition of data from intact, unfixed organs without a coverglass both in vivo and in situ.