Characterizing the 3-D field distortions in low numerical aperture fluorescence zooming microscope

In this article, we characterize the lateral field distortions in a low numerical aperture and large field-of-view (FOV) fluorescence imaging system. To this end, we study a commercial fluorescence MACROscope setup, which is a zooming microscope. The versatility of this system lies in its ability to image at different zoom ranges, so that sample preparations can be examined in three-dimensions, at cellular, organ and whole body levels. Yet, we found that the imaging system’s optics are optimized only for high magnifications where the observed FOV is small. When we studied the point-spread function (PSF) by using fluorescent polystyrene beads as “guide-stars”, we noticed that the PSF is spatially varying due to field distortions. This variation was found to be laterally symmetrical and the distortions were found to increase with the distance from the center of the FOV. In this communication, we investigate the idea of using the field at the back focal plane of an optical system for characterizing distortions. As this field is unknown, we develop a theoretical framework to retrieve the amplitude and phase of the field at the back focal pupil plane, from the empirical bead images. By using the retrieved amplitude, we can understand and characterize the underlying cause of these distortions. We also propose a few approaches, before acquisition, to either avoid it or correct it at the optical design level.