Fast label-free microscopy technique for 3D dynamic quantitative imaging of living cells

Abstract

The refractive index (RI) is an important optical characteristic that is often exploited in label-free microscopy for analysis of biological objects. A technique for 3D RI reconstruction of living cells has to be fast enough to capture the cell dynamics and preferably needs to be compatible with standard wide-field microscopes. To solve this challenging problem, we present a technique that provides fast measurement and processing of data required for real-time 3D visualization of the object RI. Specifically, the 3D RI is reconstructed from the measurement of bright-field intensity images, axially scanned by a high-speed focus tunable lens mounted in front of a sCMOS camera, by using a direct deconvolution approach designed for partially coherent light microscopy in the non-paraxial regime. Both the measurement system and the partially coherent illumination, that provides optical sectioning and speckle-noise suppression, enable compatibility with wide-field microscopes resulting in a competitive and affordable alternative to the current holographic laser microscopes. Our experimental demonstrations show video-rate 3D RI visualization of living bacteria both freely swimming and optically manipulated by using freestyle laser traps allowing for their trapping and transport along 3D trajectories. These results prove that is possible to conduct simultaneous 4D label-free quantitative imaging and optical manipulation of living cells, which is promising for the study of the cell biophysics and biology.