Multi-spectral reflection matrix for ultrafast 3D label-free microscopy

Label-free microscopy exploits light scattering to obtain a three-dimensional image of biological tissues. However, light propagation is affected by aberrations and multiple scattering, which drastically degrade the image quality and limit the penetration depth. Multi-conjugate adaptive optics and time-gated matrix approaches have been developed to compensate for aberrations but the associated frame rate is extremely limited for three-dimensional imaging. Here we develop a multi-spectral matrix approach to solve these fundamental problems. On the basis of a sparse illumination scheme and an interferometric measurement of the reflected wave field at multiple wavelengths, the focusing process can be optimized in post-processing for any voxel by addressing independently each frequency component of the reflection matrix. A proof-of-concept experiment shows a three-dimensional image of an opaque human cornea over a 0.1 mm3 field of view at a 290 nm resolution and a 1 Hz frame rate. This work paves the way towards a fully digital microscope allowing real-time, in vivo, quantitative and deep inspection of tissues. Based on the acquisition of a multi-spectral reflection matrix at a high frame rate, a fully digital microscope overcomes aberrations and multiple scattering to provide a three-dimensional image of an ex vivo opaque cornea at a resolution of 0.29 μm and 0.5 μm in the transverse and axial directions, respectively.