Spatially patterning the polarization state of a beam enables a novel approach for phase contrast microscopy, in which two foci (sample and reference) are offset along the optical axis. Polarization wavefront shaping is achieved through the addition of two custom microretarter array (RA) 25mm optics, which serve as selective converging/diverging lenses focusing right and left circular polarization components onto different focal planes. Positioning of a matched RA in the transmission path recombines the two polarization components, with phase differences manifesting and rotations in the polarization state. This approach combines benefits of Nomarski and Zernike phase contrast strategies, while minimizing common artifacts associated with each. In addition, polarization wavefront shaping provides a straightforward route for depth of field extension through addition of a single fixed 1” optic.
Conventional three-dimensional (3D) images of biological samples are typically assembled from a stack of twodimensional images acquired sequentially at different focal planes. This time-consuming manner hinders the application of 3D imaging techniques to the investigation of fast biochemical dynamics and light-sensitive biological events. The concept of multifocus imaging, which enables simultaneous acquisition of images from multiple focal planes, was introduced to achieve rapid 3D imaging. In the present study, we achieved multifocus imaging through polarization wavefront shaping via a micro-retarder array which splits the incident linearly polarized light into three beamlets that are focused to three axially-offset focal planes with ~100 μm separation. Append to an existing beam-scanning microscope, this multifocus system enables rapid 3D imaging compatible with a variety of optical microscopic approaches including laser transmittance, two-photon excited fluorescence, and second harmonic generation microscopy.
Axially-offset differential interference contrast (ADIC) microscopy was developed for quantitative phase contrast imaging (QPI) by using polarization wavefront shaping approach with a matched pair of micro-retarder arrays. In ADIC microscopy, wavefront shaping with a micro-retarder array (μRA) produces a pattern of half-wave retardance varying spatially in the azimuthal orientation of the fast-axis. For a linearly polarized input beam, the polarization pattern induced from the linearly polarized plane wave through the μRA is identical to the interference between a slightly diverging right circularly polarized (RCP) and a slightly converging left circularly polarized (LCP) plane wave. Using a 10× objective, two axially offset foci separated by 70 μm are consequently generated from the patterned wavefront with orthogonal polarization states, serving as the sample and reference focal planes respectively for QPI. A paired μRA in transmission coherently recombines the two orthogonal components to recover the incident polarization state in the absence of sample. The large spatial offset (roughly 1/10 of the field of view) between the two foci provides a stable and uniform reference. Quantitative phase contrast images are directly recovered from sample-scan measurements with a single-channel detector and lock-in amplification with fast polarization modulation. This method has been successfully used for bio-sample imaging, nanoparticle detection and refractive index calculation of silica microbeads.
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