Quantitative high dynamic range beam profiling for fluorescence microscopy

Modern developmental biology relies on optically-sectioning \uduorescence microscope techniques to produce non-destructive in-vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3-D) intensity prole of the illumination employed, the ability to directly record and analyze these proles is of great use to the uorescence microscopist or instrument builder. Though excitation beam proles can be measured indirectly using a sample of \uduorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam proling device and high dynamic range ux reconstruction algorithm that together are capable of accurately reproducing quantitative 3-D \udux maps over a large focal volume. Performance of this beam proling system is veried within an optical test bench and demonstrated for uorescence microscopy by proling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely-\udexible beam amplitude diagnostic tool for use within the life sciences