Reflection Phase Microscopy by Successive Accumulation of Interferograms

Imaging three-dimensional (3-D) structures of biological specimens without exogenous contrast agents is desired in biological and medical science in order not to disturb the physiological status of the living samples. Reflection phase microscopy based on interferometric detection has been useful for the label-free observation of such samples. However, the achievement of optical sectioning has been mainly based on the time gating set by the broad spectra of light sources. Here we propose wide-field reflection phase microscopy using a light source of narrow bandwidth, which is yet capable of achieving the optical sectioning sufficient for 3-D imaging of biological specimens. The depth selectivity is achieved by successive accumulation of interferograms (SAI) produced by synchronous angular scanning of a plane wave on both the sample and reference planes. This intensity-based cumulative process eventually results in a coherent addition of object fields that quickly attenuates the out-of-focus information along the axial direction. We theoretically investigated and numerically verified the generation of the depth selectivity by SAI. We also implemented a reflection phase microscope working with this principle and then demonstrated high-resolution 3-D imaging of living cells and small worms in a label-free manner. © 2019 American Chemical Societ