Using multimodal imaging to create 3D fluorescent images via Holographically Informed Fluorescent Imaging*

Current methods for generating 3D fluorescent images, like confocal microscopy, require scanning and significant post-processing, which can be time consuming. This is impractical for imaging live, dynamic samples. Holographic techniques, however, can produce 3D images in milliseconds using a single field of view. Localized focusing can be used to project a 3D hologram into focus at a single plane. Coregistration of the locally focused, 2D hologram with a fluorescent image of the same field of view permits the fluorescent signal to then be back-propagated to the appropriate depths within the 3D space, thereby creating a 3D fluorescent image. This multimodal imaging technique used to generate 3D fluorescent images is called Holographically Informed Fluorescent Imaging (HIFI). Simulations of HIFI using MATLAB show successful 3D reconstructions via Phase Shifting Digital Holography, combined with fluorescence imaging. A proof-of-concept experiment is underway to validate this method.