Lensfree diffractive tomography for the imaging of 3D cell cultures.

F Momey,A Berdeu,T Bordy,F Kermarrec Marcel,X Gidrol,J.-M Dinten,C Allier,N Picollet-D’Hahan

doi:10.1364/boe.7.000949

Abstract

New microscopes are needed to help realize the full potential of 3D organoid culture studies. In order to image large volumes of 3D organoid cultures while preserving the ability to catch every single cell, we propose a new imaging platform based on lensfree microscopy. We have built a lensfree diffractive tomography setup performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel and developed a dedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem. With this new imaging platform, we have been able to reconstruct a 3D volume as large as 21.5 mm (3) of a 3D organoid culture of prostatic RWPE1 cells showing the ability of these cells to assemble in 3D intricate cellular network at the mesoscopic scale. Importantly, comparisons with 2D images show that it is possible to resolve single cells isolated from the main cellular structure with our lensfree diffractive tomography setup.

Highlights

The study of in vitro cell populations remains a challenging task if one needs to gather large quantitative and systematic data over extended periods of time while preserving the integrity of the living sample
The adaptation of lensless microscopy techniques to 3D organoid cultures imaging is the scope of the present paper
The zoom in the red medallion shows the artefacts of the first method around an isolated single cell: on the xy-plane one can see white and black residues around the branches

Summary

Introduction

The study of in vitro cell populations remains a challenging task if one needs to gather large quantitative and systematic data over extended periods of time while preserving the integrity of the living sample. As discussed in Ref. 1, there is a need for a new microscopy technique that must be label-free and non-phototoxic to be as ’gentle’ as possible with the sample, and ’smart’ enough to observe the sample exhaustively at a variety of scales both in space and time. Lens-free video microscopy is addressing these needs in the context of 2D cell culture.[2, 3]. As scientists better understand the benefit of growing organoids in 3D and routinely adopt 3D culture techniques, lens-free imaging must be adapted to 3D cultures. The new challenging task is to extend lens-free microscopy techniques to the acquisitions and fully 3D reconstructions of large organoids structures.[4,5,6] The adaptation of lensless microscopy techniques to 3D organoid cultures imaging is the scope of the present paper

Methods

Results

Conclusion