Data preprocessing methods for robust Fourier ptychographic microscopy

Fourier ptychographic microscopy (FPM) is a recently proposed computational imaging technique with both high resolution and wide field-of-view. In current FP experimental setup, the dark-field images with high-angle illuminations are easily submerged by stray light and background noise due to the low signal-to-noise ratio, thus significantly degrading the reconstruction quality and also imposing a major restriction on the synthetic numerical aperture (NA) of the FP approach. To this end, an overall and systematic data preprocessing scheme for noise removal from FP's raw dataset is provided, which involves sampling analysis as well as underexposed/overexposed treatments, then followed by the elimination of unknown stray light and suppression of inevitable background noise, especially Gaussian noise and CCD dark current in our experiments. The reported non-parametric scheme facilitates great enhancements of the FP's performance, which has been demonstrated experimentally that the benefits of noise removal by these methods far outweigh its defects of concomitant signal loss. In addition, it could be flexibly cooperated with the existing state-of-the-art algorithms, producing a stronger robustness of the FP approach in various applications.Comment: 7 pages, 8 figure

In situ correction of liquid meniscus in cell culture imaging system based on parallel Fourier ptychographic microscopy (96 Eyes)

We collaborated with Amgen and spent five years in designing and fabricating next generation multi-well plate imagers based on Fourier ptychographic microscopy (FPM). A 6-well imager (Emsight) and a low-cost parallel microscopic system (96 Eyes) based on parallel FPM were reported in our previous work. However, the effect of liquid meniscus on the image quality is much stronger than anticipated, introducing obvious wavevector misalignment and additional image aberration. To this end, an adaptive wavevector correction (AWC-FPM) algorithm and a pupil recovery improvement strategy are presented to solve these challenges in situ. In addition, dual-channel fluorescence excitation is added to obtain structural information for microbiologists. Experiments are demonstrated to verify their performances. The accuracy of angular resolution with our algorithm is within 0.003 rad. Our algorithms would make the FPM algorithm more robust and practical and can be extended to other FPM-based applications to overcome similar challenges

SNR-based adaptive acquisition method for fast Fourier ptychographic microscopy

Fourier ptychographic microscopy (FPM) is a computational imaging technique with both high resolution and large field-of-view. However, the effective numerical aperture (NA) achievable with a typical LED panel is ambiguous and usually relies on the repeated tests of different illumination NAs. The imaging quality of each raw image usually depends on the visual assessments, which is subjective and inaccurate especially for those dark field images. Moreover, the acquisition process is really time-consuming.In this paper, we propose a SNR-based adaptive acquisition method for quantitative evaluation and adaptive collection of each raw image according to the signal-to-noise ration (SNR) value, to improve the FPM's acquisition efficiency and automatically obtain the maximum achievable NA, reducing the time of collection, storage and subsequent calculation. The widely used EPRY-FPM algorithm is applied without adding any algorithm complexity and computational burden. The performance has been demonstrated in both USAF targets and biological samples with different imaging sensors respectively, which have either Poisson or Gaussian noises model. Further combined with the sparse LEDs strategy, the number of collection images can be shorten to around 25 frames while the former needs 361 images, the reduction ratio can reach over 90%. This method will make FPM more practical and automatic, and can also be used in different configurations of FPM.Comment: 11 pages, 6 figure

Illumination coding meets uncertainty learning: toward reliable AI-augmented phase imaging

We propose a physics-assisted deep learning (DL) framework for large space-bandwidth product (SBP) phase imaging. We design an asymmetric coded illumination scheme to encode high-resolution phase information across a wide field-of-view. We then develop a matching DL algorithm to provide large-SBP phase estimation. We show that this illumination coding scheme is highly scalable in achieving flexible resolution, and robust to experimental variations. We demonstrate this technique on both static and dynamic biological samples, and show that it can reliably achieve 5X resolution enhancement across 4X FOVs using only five multiplexed measurements -- more than 10X data reduction over the state-of-the-art. Typical DL algorithms tend to provide over-confident predictions, whose errors are only discovered in hindsight. We develop an uncertainty learning framework to overcome this limitation and provide predictive assessment to the reliability of the DL prediction. We show that the predicted uncertainty maps can be used as a surrogate to the true error. We validate the robustness of our technique by analyzing the model uncertainty. We quantify the effect of noise, model errors, incomplete training data, and "out-of-distribution" testing data by assessing the data uncertainty. We further demonstrate that the predicted credibility maps allow identifying spatially and temporally rare biological events. Our technique enables scalable AI-augmented large-SBP phase imaging with dependable predictions.Published versio

Deep learning approach to Fourier ptychographic microscopy

Convolutional neural networks (CNNs) have gained tremendous success in solving complex inverse problems. The aim of this work is to develop a novel CNN framework to reconstruct video sequences of dynamic live cells captured using a computational microscopy technique, Fourier ptychographic microscopy (FPM). The unique feature of the FPM is its capability to reconstruct images with both wide field-of-view (FOV) and high resolution, i.e. a large space-bandwidth-product (SBP), by taking a series of low resolution intensity images. For live cell imaging, a single FPM frame contains thousands of cell samples with different morphological features. Our idea is to fully exploit the statistical information provided by these large spatial ensembles so as to make predictions in a sequential measurement, without using any additional temporal dataset. Specifically, we show that it is possible to reconstruct high-SBP dynamic cell videos by a CNN trained only on the first FPM dataset captured at the beginning of a time-series experiment. Our CNN approach reconstructs a 12800×10800 pixel phase image using only ∼25 seconds, a 50× speedup compared to the model-based FPM algorithm. In addition, the CNN further reduces the required number of images in each time frame by ∼ 6×. Overall, this significantly improves the imaging throughput by reducing both the acquisition and computational times. The proposed CNN is based on the conditional generative adversarial network (cGAN) framework. We further propose a mixed loss function that combines the standard image domain loss and a weighted Fourier domain loss, which leads to improved reconstruction of the high frequency information. Additionally, we also exploit transfer learning so that our pre-trained CNN can be further optimized to image other cell types. Our technique demonstrates a promising deep learning approach to continuously monitor large live-cell populations over an extended time and gather useful spatial and temporal information with sub-cellular resolution.We would like to thank NVIDIA Corporation for supporting us with the GeForce Titan Xp through the GPU Grant Program. (NVIDIA Corporation; GeForce Titan Xp through the GPU Grant Program)First author draf

Iterative Phase Retrieval Algorithms for Scanning Transmission Electron Microscopy

Scanning transmission electron microscopy (STEM) has been extensively used for imaging complex materials down to atomic resolution. The most commonly employed STEM imaging modality of annular dark field produces easily-interpretable contrast, but is dose-inefficient and produces little to no contrast for light elements and weakly-scattering samples. An alternative is to use phase contrast STEM imaging, enabled by high speed detectors able to record full images of a diffracted STEM probe over a grid of scan positions. Phase contrast imaging in STEM is highly dose-efficient, able to measure the structure of beam-sensitive materials and even biological samples. Here, we comprehensively describe the theoretical background, algorithmic implementation details, and perform both simulated and experimental tests for three iterative phase retrieval STEM methods: focused-probe differential phase contrast, defocused-probe parallax imaging, and a generalized ptychographic gradient descent method implemented in two and three dimensions. We discuss the strengths and weaknesses of each of these approaches using a consistent framework to allow for easier comparison. This presentation of STEM phase retrieval methods will make these methods more approachable, reproducible and more readily adoptable for many classes of samples.Comment: 25 pages, 11 figures, 1 tabl

Deep learning approach to Fourier ptychographic microscopy

Convolutional neural networks (CNNs) have gained tremendous success in solving complex inverse problems. The aim of this work is to develop a novel CNN framework to reconstruct video sequence of dynamic live cells captured using a computational microscopy technique, Fourier ptychographic microscopy (FPM). The unique feature of the FPM is its capability to reconstruct images with both wide field-of-view (FOV) and high resolution, i.e. a large space-bandwidth-product (SBP), by taking a series of low resolution intensity images. For live cell imaging, a single FPM frame contains thousands of cell samples with different morphological features. Our idea is to fully exploit the statistical information provided by this large spatial ensemble so as to make predictions in a sequential measurement, without using any additional temporal dataset. Specifically, we show that it is possible to reconstruct high-SBP dynamic cell videos by a CNN trained only on the first FPM dataset captured at the beginning of a time-series experiment. Our CNN approach reconstructs a 12800X10800 pixels phase image using only ~25 seconds, a 50X speedup compared to the model-based FPM algorithm. In addition, the CNN further reduces the required number of images in each time frame by ~6X. Overall, this significantly improves the imaging throughput by reducing both the acquisition and computational times. The proposed CNN is based on the conditional generative adversarial network (cGAN) framework. Additionally, we also exploit transfer learning so that our pre-trained CNN can be further optimized to image other cell types. Our technique demonstrates a promising deep learning approach to continuously monitor large live-cell populations over an extended time and gather useful spatial and temporal information with sub-cellular resolution

In situ correction of liquid meniscus in cell culture imaging system based on parallel Fourier ptychographic microscopy (96 Eyes)

We collaborated with Amgen and spent five years in designing and fabricating next generation multi-well plate imagers based on Fourier ptychographic microscopy (FPM). A 6-well imager (Emsight) and a low-cost parallel microscopic system (96 Eyes) based on parallel FPM were reported in our previous work. However, the effect of liquid meniscus on the image quality is much stronger than anticipated, introducing obvious wavevector misalignment and additional image aberration. To this end, an adaptive wavevector correction (AWC-FPM) algorithm and a pupil recovery improvement strategy are presented to solve these challenges in situ. In addition, dual-channel fluorescence excitation is added to obtain structural information for microbiologists. Experiments are demonstrated to verify their performances. The accuracy of angular resolution with our algorithm is within 0.003 rad. Our algorithms would make the FPM algorithm more robust and practical and can be extended to other FPM-based applications to overcome similar challenges

py4DSTEM: a software package for multimodal analysis of four-dimensional scanning transmission electron microscopy datasets

Scanning transmission electron microscopy (STEM) allows for imaging, diffraction, and spectroscopy of materials on length scales ranging from microns to atoms. By using a high-speed, direct electron detector, it is now possible to record a full 2D image of the diffracted electron beam at each probe position, typically a 2D grid of probe positions. These 4D-STEM datasets are rich in information, including signatures of the local structure, orientation, deformation, electromagnetic fields and other sample-dependent properties. However, extracting this information requires complex analysis pipelines, from data wrangling to calibration to analysis to visualization, all while maintaining robustness against imaging distortions and artifacts. In this paper, we present py4DSTEM, an analysis toolkit for measuring material properties from 4D-STEM datasets, written in the Python language and released with an open source license. We describe the algorithmic steps for dataset calibration and various 4D-STEM property measurements in detail, and present results from several experimental datasets. We have also implemented a simple and universal file format appropriate for electron microscopy data in py4DSTEM, which uses the open source HDF5 standard. We hope this tool will benefit the research community, helps to move the developing standards for data and computational methods in electron microscopy, and invite the community to contribute to this ongoing, fully open-source project