We present a diffraction-phase and fluorescence 3D microscope as novel bimodal imaging technique, which provides simultaneous phase and multi-color epi-fluorescence acquisitions of living multicellular samples. The instrument consists of an LED-array to acquire intensity images at different illumination angles and an epifluorescence setup for fluorescence excitation. The 3D sample’s optical properties are reconstructed using the beam propagation method embedded inside a deep learning framework. To obtain the fluorescence reconstructions, we developed a novel incoherent model that takes into account the heterogeneous refractive indexes of the scattering sample. We validated the technique on long-term acquisitions of mouse embryos and 3D liver organoids under physiological conditions.
One of the directions of development in quantitative phase imaging is to provide the capability to reconstruct the phase or preferably refractive index (RI) distribution within thick, highly scattering samples. This direction coincides with current trends in biology, where three-dimensional (3D) organoids are currently replacing standard 2D cultures as more physiological models for tissue growth and organ formation in a dish. The biological complexity of these 3D structures makes the imaging and RI reconstruction particularly challenging, and thus calibration as well as validation structures are important and sought-after tools in instrumentation development. For this reason, in this work, we present the full preparation and measurement procedure for organoid phantoms printed with two-photon polymerization along with the method to obtain the ground truth of the object structure independently of RI reconstruction errors and artifacts.
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