Neural network‐assisted localization of clustered point spread functions in single‐molecule localization microscopy

Single‐molecule localization microscopy (SMLM), which has revolutionized nanoscale imaging, faces challenges in densely labelled samples due to fluorophore clustering, leading to compromised localization accuracy. In this paper, we propose a novel convolutional neural network (CNN)‐assisted approach to address the issue of locating the clustered fluorophores. Our CNN is trained on a diverse data set of simulated SMLM images where it learns to predict point spread function (PSF) locations by generating Gaussian blobs as output. Through rigorous evaluation, we demonstrate significant improvements in PSF localization accuracy, especially in densely labelled samples where traditional methods struggle. In addition, we employ blob detection as a post‐processing technique to refine the predicted PSF locations and enhance localization precision. Our study underscores the efficacy of CNN in addressing clustering challenges in SMLM, thereby advancing spatial resolution and enabling deeper insights into complex biological structures.