Abstract
We present a new Phase-Retrieved Tomography (PRT) method to radically improve mesoscopic imaging at regimes beyond one transport mean-free-path and achieve high resolution, uniformly throughout the volume of opaque samples. The method exploits multi-view acquisition in a hybrid Selective Plane Illumination Microscope (SPIM) and Optical Projection Tomography (OPT) setup and a three-dimensional Gerchberg-Saxton phase-retrieval algorithm applied in 3D through the autocorrelation sinogram. We have successfully applied this innovative protocol to image optically dense 3D cell cultures in the form of tumor spheroids, highly versatile models to study cancer behavior and response to chemotherapy. We have thus achieved a significant improvement of resolution in depths not yet accessible with the currently used methods in SPIM/OPT, while overcoming all registration and alignment problems inherent to these techniques.
Highlights
The use of light for visualizing biological processes in living organisms has been a foundational paradigm for biology for almost four centuries, since the invention of the microscope
Because of the novel use of the algorithm combined with Selective Plane Illumination Microscopy (SPIM)/Optical Projection Tomography (OPT), we found appropriate referring to this approach with the name Phase-Retrieved Tomography (PRT)
A schematic of the experimental measurements performed in a combined SPIM/OPT setup is illustrated in Fig. 1: per each rotation angle, we acquired a collection of tomographic slices by illuminating the sample with a light sheet perpendicular to the camera plane
Summary
The use of light for visualizing biological processes in living organisms has been a foundational paradigm for biology for almost four centuries, since the invention of the microscope. Tomographic principles are conceptually simple and well described and characterized, optically sectioning the sample for internal functional and structural inspection often requires a tremendous effort on both computational and experimental aspects in order to achieve a satisfying image quality This becomes even more relevant and demanding, in regimes at around one transport mean free path and higher, where strong light scattering restricts high-resolution imaging in the macroscopic scales. Different camera images are recorded, either by scanning the sample by changing the position of the light sheet or translating the specimen throughout a fixed single plane illumination In such a way, the tomographic volume is built with very little computational effort, in cases of optically transparent or chemically cleared specimens. Because of the novel use of the algorithm combined with SPIM/OPT, we found appropriate referring to this approach with the name Phase-Retrieved Tomography (PRT)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
