Optimal illumination scheme for isotropic quantitative differential phase contrast microscopy

Differential phase contrast microscopy (DPC) provides high-resolution quantitative phase distribution of thin transparent samples under multi-axis asymmetric illuminations. Typically, illumination in DPC microscopic systems is designed with 2-axis half-circle amplitude patterns, which, however, result in a non-isotropic phase contrast transfer function (PTF). Efforts have been made to achieve isotropic DPC by replacing the conventional half-circle illumination aperture with radially asymmetric patterns with 3-axis illumination or gradient amplitude patterns with 2-axis illumination. Nevertheless, these illumination apertures were empirically designed based on empirical criteria related to the shape of the PTF, leaving the underlying theoretical mechanisms unexplored. Furthermore, the frequency responses of the PTFs under these engineered illuminations have not been fully optimized, leading to suboptimal phase contrast and signal-to-noise ratio (SNR) for phase reconstruction. In this Letter, we provide a rigorous theoretical analysis about the necessary and sufficient conditions for DPC to achieve perfectly isotropic PTF. In addition, we derive the optimal illumination scheme to maximize the frequency response for both low and high frequencies (from 0 to 2N Aob j), and meanwhile achieve perfectly isotropic PTF with only 2-axis intensity measurements. We present the derivation, implementation, simulation and experimental results demonstrating the superiority of our method over state-of-the-arts in both phase reconstruction accuracy and noise-robustness.https://arxiv.org/abs/1903.10718Accepted manuscrip

High-resolution transport-of-intensity quantitative phase microscopy with annular illumination

For quantitative phase imaging (QPI) based on transport-of-intensity equation (TIE), partially coherent illumination provides speckle-free imaging, compatibility with brightfield microscopy, and transverse resolution beyond coherent diffraction limit. Unfortunately, in a conventional microscope with circular illumination aperture, partial coherence tends to diminish the phase contrast, exacerbating the inherent noise-to-resolution tradeoff in TIE imaging, resulting in strong low-frequency artifacts and compromised imaging resolution. Here, we demonstrate how these issues can be effectively addressed by replacing the conventional circular illumination aperture with an annular one. The matched annular illumination not only strongly boosts the phase contrast for low spatial frequencies, but significantly improves the practical imaging resolution to near the incoherent diffraction limit. By incorporating high-numerical aperture (NA) illumination as well as high-NA objective, it is shown, for the first time, that TIE phase imaging can achieve a transverse resolution up to 208 nm, corresponding to an effective NA of 2.66. Time-lapse imaging of in vitro Hela cells revealing cellular morphology and subcellular dynamics during cells mitosis and apoptosis is exemplified. Given its capability for high-resolution QPI as well as the compatibility with widely available brightfield microscopy hardware, the proposed approach is expected to be adopted by the wider biology and medicine community.Comment: This manuscript was originally submitted on 20 Feb. 201

Optimal illumination scheme for isotropic quantitative differential phase contrast microscopy

Differential phase contrast microscopy (DPC) provides high-resolution quantitative phase distribution of thin transparent samples under multi-axis asymmetric illuminations. Typically, illumination in DPC microscopic systems is designed with 2-axis half-circle amplitude patterns, which, however, result in a non-isotropic phase contrast transfer function (PTF). Efforts have been made to achieve isotropic DPC by replacing the conventional half-circle illumination aperture with radially asymmetric patterns with 3-axis illumination or gradient amplitude patterns with 2-axis illumination. Nevertheless, these illumination apertures were empirically designed based on empirical criteria related to the shape of the PTF, leaving the underlying theoretical mechanisms unexplored. Furthermore, the frequency responses of the PTFs under these engineered illuminations have not been fully optimized, leading to suboptimal phase contrast and signal-to-noise ratio (SNR) for phase reconstruction. In this Letter, we provide a rigorous theoretical analysis about the necessary and sufficient conditions for DPC to achieve perfectly isotropic PTF. In addition, we derive the optimal illumination scheme to maximize the frequency response for both low and high frequencies (from 0 to 2NAobj), and meanwhile achieve perfectly isotropic PTF with only 2-axis intensity measurements. We present the derivation, implementation, simulation and experimental results demonstrating the superiority of our method over state-of-the-arts in both phase reconstruction accuracy and noise-robustness.Comment: 18 pages, 9 figure

Quantitative Phase Imaging Camera With a Weak Diffuser

We introduce the quantitative phase imaging camera with a weak diffuser (QPICWD) as an effective scheme of quantitative phase imaging (QPI) based on normal microscope platforms. The QPICWD is an independent compact camera measuring object induced phase delay under low-coherence quasi-monochromatic illumination by examining the deformation of the speckle intensity pattern. By interpreting the speckle deformation with an ensemble average of the geometric flow, we can obtain the high-resolution distortion field via the transport of intensity equation (TIE). Since the phase measured by TIE is the generalized phase of the partially coherent image, rather than the phase of the measured object, we analyze the effect of illumination coherence and imaging numerical aperture (NA) on the accuracy of phase retrieval, revealing that the sample's phase can be reliably reconstructed under the conditions that the coherence parameter (the ratio of illumination NA to objective NA) of the Köhler illumination is between 0.3 and 0.5. We present some applications for the proposed design involving nondestructive optical testing of microlens array with nanometric thickness and imaging of fixed and live unstained HeLa cells. Since the designed QPI camera does not require any modification of the widely available bright-field microscope or additional accessories for its use, it is expected to be applied by the broader communities of biology and medicine