Homographically generated light sheets for the microscopy of large specimens

We compare the performance of linear and nonlinear methods for aligning the excitation and detection planes throughout volumes of large specimens in digitally scanned light sheet microscopy. An effective nonlinear method involves the registration of four corner extrema of the imaging volume via a projective transform. We show that this improves the light collection efficiency of the commonly used three-point affine registration by an average of 42% over a typical specimen volume. Accurate illumination/detection registration methods are now pertinent to biological research in view of current trends towards imaging large or expanded samples, at depth, with diffraction limited resolution.