Measuring the point spread function of a wide-field fluorescence microscope

The point spread function (PSF) of a wide-field fluorescence microscope, which measures the system's impulse response, is a crucial parameter in non-blind deconvolution. To determine the PSF, traditional methods treat a fluorescent bead as a point source whose optical field distribution is approximate to it. However, beads with sufficiently small sizes are often difficult to observe in a microscope due to their low brightness. In this paper, we present a new approach to measure the PSF under the condition of non-ideal point sources and low signal-to-noise ratio (SNR). We first recorded a focal stack of fluorescent beads and automatically selected those that met certain requirements. Then, we computed a two-dimensional (2D) PSF for each plane at different defocus distances, some of which were fitted according to Gaussian distribution and the rest were calculated mainly by averaging the beads. Finally, we combined each 2D PSF based on the energy distribution to obtain a three-dimensional (3D) PSF. The proposed algorithm has been tested on the Real-time, Ultra-large-Scale imaging at High-resolution (RUSH) macroscope. By implementing deconvolution using the PSF derived by this method and a traditional method respectively, results show that the proposed algorithm has achieved a more accurate measurement of the PSF.