FRC-QE: a robust and comparable 3D microscopy image quality metric for cleared organoids

Three-dimensional stem-cell-derived organoids are a powerful tool for studying cellular processes in tissue-like structures, enabling in vitro experiments in an organ-specific context. While organoid research has been closely linked to advances in fluorescence microscopy, capturing cellular structures within their global context in an organoid often remains challenging due to the organoid’s dense structure and opacity. The development of optical clearing methods has provided a solution for fixed organoids but optimizing clearing protocols for a given sample type and staining can be challenging. Importantly, quantitative measures for assessing image quality throughout cleared fluorescent samples are missing. Here, we propose Fourier ring correlation quality estimation (FRC-QE) as a new metric for automated 3D image quality estimation in cleared organoids. We show that FRC-QE robustly captures differences in clearing efficiency within an organoid, across replicates and clearing protocols, as well as for different microscopy modalities. FRC-QE is open-source, written in ImgLib2 and provided as an easy-to-use and macro-scriptable plugin for the popular Fiji software. We therefore envision FRC-QE to fill the gap of providing a reliable quality metric for testing, optimizing and comparing optical clearing methods.