No abstract

High-throughput quantitative phase imaging (QPI) is essential to cellular phenotypes characterization as it allows high-content cell analysis and avoids adverse effects of staining reagents on cellular viability and cell signaling. Among different approaches, Fourier ptychographic microscopy (FPM) is probably the most promising technique to realize high-throughput QPI by synthesizing a wide-field, high-resolution complex image from multiple angle-variably illuminated, low-resolution images. However, the large dataset requirement in conventional FPM significantly limits its imaging speed, resulting in low temporal throughput. Moreover, the underlying theoretical mechanism as well as optimum illumination scheme for high-accuracy phase imaging in FPM remains unclear. Herein, we report a high-speed FPM technique based on programmable annular illuminations (AIFPM). The optical-transfer-function (OTF) analysis of FPM reveals that the low-frequency phase information can only be correctly recovered if the LEDs are precisely located at the edge of the objective numerical aperture (NA) in the frequency space. By using only 4 low-resolution images corresponding to 4 tilted illuminations matching a 10×, 0.4 NA objective, we present the high-speed imaging results of in vitro Hela cells mitosis and apoptosis at a frame rate of 25 Hz with a full-pitch resolution of 655 nm at a wavelength of 525 nm (effective NA = 0.8) across a wide field-of-view (FOV) of 1.77 mm2, corresponding to a space–bandwidth–time product of 411 megapixels per second. Our work reveals an important capability of FPM towards high-speed high-throughput imaging of in vitro live cells, achieving video-rate QPI performance across a wide range of scales, both spatial and temporal.

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 A ob 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.

Computational microscopy, as a subfield of computational imaging, combines optical manipulation and image algorithmic reconstruction to recover multi-dimensional microscopic images or information of micro-objects. In recent years, the revolution in light-emitting diodes (LEDs), low-cost consumer image sensors, modern digital computers, and smartphones provide fertile opportunities for the rapid development of computational microscopy. Consequently, diverse forms of computational microscopy have been invented, including digital holographic microscopy (DHM), transport of intensity equation (TIE), differential phase contrast (DPC) microscopy, lens-free on-chip holography, and Fourier ptychographic microscopy (FPM). These computational microscopy techniques not only provide high-resolution, label-free, quantitative phase imaging capability but also decipher new and advanced biomedical research and industrial applications. Nevertheless, most computational microscopy techniques are still at an early stage of “proof of concept” or “proof of prototype” (based on commercially available microscope platforms). Translating those concepts to stand-alone optical instruments for practical use is an essential step for the promotion and adoption of computational microscopy by the wider bio-medicine, industry, and education community. In this paper, we present four smart computational light microscopes (SCLMs) developed by our laboratory, i.e., smart computational imaging laboratory (SCILab) of Nanjing University of Science and Technology (NJUST), China. These microscopes are empowered by advanced computational microscopy techniques, including digital holography, TIE, DPC, lensless holography, and FPM, which not only enables multi-modal contrast-enhanced observations for unstained specimens, but also can recover their three-dimensional profiles quantitatively. We introduce their basic principles, hardware configurations, reconstruction algorithms, and software design, quantify their imaging performance, and illustrate their typical applications for cell analysis, medical diagnosis, and microlens characterization.

We present a single-shot quantitative phase imaging (QPI) method based on color-multiplexed Fourier ptychographic microscopy (FPM). Three light-emitting diode (LED) elements with respective R/G/B channels in a programmable LED array illuminate the specimen simultaneously, providing triangle oblique illuminations matching the numerical aperture of the objective precisely. A color image sensor records the light transmitted through the specimen, and three monochromatic intensity images at each color channel are then separated and utilized to recover the phase of the specimen. After one-step deconvolution based on the phase contrast transfer function, the obtained initial phase map is further refined by the FPM-based iterative recovery algorithm to overcome pixel-aliasing and improve the phase recovery accuracy. The high-speed, high-throughput QPI capabilities of the proposed approach are demonstrated by imaging HeLa cells mitosis in vitro, achieving a half-pitch resolution of 388 nm across a wide field of view of 1.33  mm at camera-limited frame rates (50 fps).