High-speed Fourier ptychographic microscopy based on programmable annular illuminations

Fourier ptychographic microscopy (FPM) is a newly developed microscopic technique for large field of view, high-resolution and quantitative phase imaging by combining the techniques from ptychographic imaging, aperture synthesizing and phase retrieval. In FPM, an LED array is utilized to illuminate the specimen from different angles and the corresponding intensity images are synthesized to reconstruct a high-resolution complex field. As a flexible and low-cost approach to achieve high-resolution, wide-field and quantitative phase imaging, FPM is of enormous potential in biomedical applications such as hematology and pathology. Conventionally, the FPM reconstruction problem is solved by using a phase retrieval method, termed Alternate Projection. By iteratively updating the Fourier spectrum with low-resolution-intensity images, the result converges to a high-resolution complex field. Here we propose a new FPM reconstruction framework with deep learning methods and design a multiscale, deep residual neural network for FPM reconstruction. We employ the widely used open-source deep learning library PyTorch to train and test our model and carefully choose the hyperparameters of our model. To train and analyze our model, we build a large-scale simulation dataset with an FPM imaging model and an actual dataset captured with an FPM system. The simulation dataset and actual dataset are separated as training datasets and test datasets, respectively. Our model is trained with the simulation training dataset and fine tuned with the fine-tune dataset, which contains actual training data. Both our model and the conventional method are tested on the simulation test dataset and the actual test dataset to evaluate the performances. We also show the reconstruction result of another neural network-based method for comparison. The experiments demonstrate that our model achieves better reconstruction results and consumes much less time than conventional methods. The results also point out that our model is more robust under system aberrations such as noise and blurring (fewer intensity images) compared with conventional methods.

As a typical computational method, Fourier ptychographic microscopy (FPM) can realize high spatial resolution and quantitative phase imaging while preserving the large field of view with a low numerical aperture (NA) objective. A programmable light-emitting diode (LED) array is used as a typical illuminator in an FPM system, and the illumination parameters of each LED element are crucial to the success of the FPM reconstruction algorithm. Compared with LED arrays arranged in rectangular arrays, LED arrays with special structures such as domes or rings can effectively improve FPM imaging results and imaging efficiency. As a trade-off, their calibration difficulty is greatly increased due to the lack of geometric constraints of rectangular arrays. In this paper, we propose an effective hybrid full-pose parameter calibration method for freeform LED array illuminators, combining stereoscopic 3D imaging techniques and the geometric constraints of the microscopic platform. First, a stereovision system is used to obtain the accurate 3D position of each LED element of the freeform illuminator and to construct a rigid 3D coordinate LED array system. Then, calibration between the coordinate system of the LED array and that of the optical imaging component is realized according to the geometric features of the brightfield-to-darkfield edges. Finally, we verify the feasibility and effectiveness of the proposed method through full-pose parameter calibration of LED arrays with different arrangement rules.

White blood cells (WBC) play a significant role in human immune system, so WBC detection is a meaningful work. In this paper, we propose a novel framework which combines Fourier ptychographic microscopy (FPM) and SO-you only look once (YOLO) for WCB detection. FPM is a recently developed microscope technology which can achieve high-resolution, wide-field, and quantitative phase imaging at the same time without mechanical moving and phase measurement. With the FPM, we can get high resolution, wide field-of-view blood cell images at one time. After obtaining high-resolution images, we propose SO-YOLO as our detection method. As a convolutional neural network, the SO-YOLO outperforms the state-of-the-art detection methods both in accuracy and speed. The proposed framework in this paper can be used in clinical diagnose, because the experiment setup is flexible and the detection is fast. The data set in this paper is made with FPM by ourselves. Our data set contains 1000 color high resolution images and all the WBC has been detected and counted by a human expert. The detection results by the experts are regarded as the ground truth. The experiment results show that the SO-YOLO performs well in detecting small objects compared with other methods.

Fourier ptychography microscopy(FPM) is a recently developed computational imaging approach for microscopic super-resolution imaging. By turning on each light-emitting-diode (LED) located on different position on the LED array sequentially and acquiring the corresponding images that contain different spatial frequency components, high spatial resolution and quantitative phase imaging can be achieved in the case of large field-of-view. Nevertheless, FPM has high requirements for the system construction and data acquisition processes, such as precise LEDs position, accurate focusing and appropriate exposure time, which brings many limitations to its practical applications. In this paper, inspired by artificial neural network, we propose a Fourier ptychography multi-parameter neural network (FPMN) with composite physical prior optimization. A hybrid parameter determination strategy combining physical imaging model and data-driven network training is proposed to recover the multi layers of the network corresponding to different physical parameters, including sample complex function, system pupil function, defocus distance, LED array position deviation and illumination intensity fluctuation, etc. Among these parameters, LED array position deviation is recovered based on the features of brightfield to darkfield transition low-resolution images while the others are recovered in the process of training of the neural network. The feasibility and effectiveness of FPMN are verified through simulations and actual experiments. Therefore FPMN can evidently reduce the requirement for practical applications of FPM.

Adaptive denoising method for Fourier ptychographic microscopy

Structural imaging using light microscopy is a cornerstone of histology and cytology. However, the utility of the optical microscope for diagnostic imaging is limited by the fundamental tradeoff between the field of view and spatial resolution and a reliance on exogenous dyes to generate sufficient image contrast. Fourier Ptychographic Microscopy (FPM) is a complex imaging modality with the potential to overcome these limitations by recovering high-resolution images of sample amplitude and phase from a set of low-resolution raw images captured under inclined illumination. In this article we explore the application of FPM to clinical imaging using a simple, low-cost FPM system and simulated and experimental data to explore the influence of both image acquisition parameters and hardware configuration on image quality and imaging throughput. The practical performance of the method is investigated by imaging peripheral blood films and histological tissue sections. We find that, at the cost of increased computational complexity, FPM increases the information capture capacity of the optical microscope significantly, allowing label-free examination and quantification of features such as tissue and cell morphology over large sample areas.

Pushing the resolution limit to the nanoscale is a critical challenge for applying the reflective Fourier ptychographic microscopy (FPM) to metrologies for characterization of nanoscale features. Characterization of opaque nanoscale samples using reflective FPM requires chiefly a light source with shorter wavelength to obtain nanoscale resolution, as state-of-the-art device sizes have become sub-100 nm or deep sub-wavelength. We report a reflective deep ultraviolet (DUV) FPM featured by an aperture scanning illumination based on the epi-illumination scheme for accommodating a 193 nm excimer laser source and a high numerical aperture (NA) catadioptric objective lens. The illumination system enables robust control of high-NA angular illumination and optimal energy fluence for FPM imaging and prevention of damage to the sample and optical components. The implemented reflective DUV FPM demonstrated image reconstruction of multiline targets with a minimum linewidth of 80 nm with an average contrast six times higher than conventional DUV microscopy.

Fourier Ptychographic Microscopy (FPM) and Differential Phase Contrast (DPC) are quantitative phase imaging (QPI) methods that recover the complex transmittance function of a sample through coded illumination measurements and phase retrieval optimization. The successes of these methods rely upon acquiring several or possibly hundreds of illumination-encoded measurements. The multi-shot nature of such methods limits their temporal resolution. Similar to motion-induced blur during a long photographic exposure, motion occurring during these acquisitions causes spatial distortion and errors in the reconstructed phase, which inhibits these methods' ability to image fast moving live samples. Here we present a novel approach to correct for motion during QPI capture that relies on motion navigation to register measurements together prior to phase retrieval. The different illumination patterns required for QPI cause the measurements to have a different contrasts. This makes it difficult to use standard registration approaches to estimate complex sample motion directly from the measurements. Instead, we use a color-multiplexed navigator signal (red) that is comprised of a constant illumination pattern and leverage a color camera to separate it from the primary QPI information (green). The reliable motion estimate allows measurements to be shared across time points through image registration. This enables a full set of measurements for a phase retrieval problem to be solved at each time point. We demonstrate proof-of-concept experimental results in which blurring due to live sample motion (swimming Zebra fish, cell motion, and organelle movement) is reduced.

Fourier Ptychographic Microscopy (FPM) is a computational imaging method that is able to super-resolve features beyond the diffraction-limit set by the objective lens of a traditional microscope. This is accomplished by using synthetic aperture and phase retrieval algorithms to combine many measurements captured by an LED array microscope with programmable source patterns. FPM provides simultaneous large field-of-view and high resolution imaging, but at the cost of reduced temporal resolution, thereby limiting live cell applications. In this work, we learn LED source pattern designs that compress the many required measurements into only a few, with negligible loss in reconstruction quality or resolution. This is accomplished by recasting the super-resolution reconstruction as a Physics-based Neural Network and learning the experimental design to optimize the network’s overall performance. Specifically, we learn LED patterns for different applications (e.g. amplitude contrast and quantitative phase imaging) and show that the designs we learn through simulation generalize well in the experimental setting. Further, we discuss a context-specific loss function, practical memory limitations, and interpretability of our learned designs.

Deep neural network (DNN) models, particularly convolutional neural networks (CNNs), have demonstrated remarkable performance in biomedical image classification due to their ability to automatically learn features from large datasets. One common challenge in the preparation of large, microscopic datasets for DNN tasks is sample defocusing, potentially impairing the model performance. To handle defocusing, computational imaging, or specifically quantitative phase imaging (QPI), performs digital refocusing by using both the phase and the amplitude of the complex optical field. This leads us to investigate whether feeding the complex field into DNN would potentially address the defocusing problem as in-focus information is implicitly encoded in the complex field. In this paper, we assess the feasibility of employing neural networks to directly process full amplitude and phase data from a defocus plane without digital refocusing. Our specific focus lies in understanding the tolerance for defocus in image classification neural networks when amplitude and phase are taken as inputs. To accomplish this, we used Fourier ptychography microscopy (FPM) to acquire in-focus phase and amplitude images of two distinct object types - normal red blood cells and echinocytes. We then digitally propagate the complex field to generate progressively defocused images of the samples to serve as training and testing datasets for image classification neural networks. While the digitally defocused images contain sufficient information to recover the original in-focus images, we observed that current standard implementations of deep learning models are unable to effectively utilize the defocused field to distinguish between the two cell types. We conclude that the physical-model-based digital refocusing capability of QPI remains indispensable for overcoming defocusing issues in current standard DNN models.

Existing quantitative phase imaging (QPI) techniques are faced with an inherent trade-off between phase imaging fidelity and temporal resolution. Here, we propose a general algorithmic framework for QPI reconstruction that enables frame-rate-limited holographic imaging. It takes an inverse problem approach by formulating phase retrieval as a nonsmooth nonconvex optimization problem. Efficient solvers for the problem are derived whose algorithmic behaviors have been studied from both theoretical and experimental perspectives. The proposed framework is applicable to various existing holographic imaging configurations, and makes it possible to incorporate advanced image priors for quality enhancement.

In this research, the accuracy of image classification with Fourier Ptychography Microscopy (FPM) has been systematically investigated. Multiple linear regression shows a strong linear relationship between the results of image classification accuracy and image visual appearance quality based on PSNR and SSIM with multiple training datasets including MINST, Fashion MNIST, Cifar, Caltech 101, and customized training datasets. It is, therefore, feasible to predict the image classification accuracy only based on PSNR and SSIM. It is also found that the image classification accuracy of FPM reconstructed with higher resolution images is significantly different from using the lower resolution images under the lower numerical aperture (NA) condition. The difference is yet less pronounced under the higher NA condition.

Fourier ptychographic microscopy (FPM) is an attractive method to extend the resolution beyond the conventional limit defined by a microscope optics, sharing properties with ptychographic, synthetic aperture imaging and phase retrieval. The algorithm uses a sequence of low-resolution (LR) images acquired under angularly varying illumination to reconstruct a high-resolution (HR) image. However, traditional FPM may trap to the sub-optimal solution, since brute-force replacement in the Fourier domain is applied. To address this problem, we propose here a weighted replacement for Fourier ptychographic microscopy (WFPM). We employ the weighted average of spectrums corresponding to different illumination angle to replace the overlapped regions in the Fourier domain. A series of experimental results demonstrate that the reconstructed image using the proposed WFPM shows a better quality and a faster convergence compared with the results obtained by FPM.

We propose a physics-assisted deep neural network scheme in Fourier ptychographic microscopy (FPM) using untrained deep neural network priors (FPMUP) to achieve a high-resolution image reconstruction from multiple low-resolution images. Unlike the traditional training type of deep neural network that requires a large labelled dataset, this proposed scheme does not require training and instead outputs the high-resolution image by optimizing the parameters of neural networks to fit the experimentally measured low-resolution images. Besides the amplitude and phase of the sample function, another two parallel neural networks that generate the general pupil function and illumination intensity factors are incorporated into the carefully designed neural networks, which effectively improves the image quality and robustness when both the aberration and illumination intensity fluctuation are present in FPM. Reconstructions using simulated and experimental datasets are demonstrated, showing that the FPMUP scheme has better image quality than the traditional iterative algorithms, especially for the phase recovery, but at the expense of increasing computational cost. Most importantly, it is found that the FPMUP scheme can predict the Fourier spectrum of the sample outside synthetic aperture of FPM and thus eliminate the ringing effect of the recovered images due to the spectral truncation. Inspired by deep image prior in the field of image processing, we may impute the expansion of Fourier spectrums to the deep prior rooted in the architecture of the careful designed four parallel deep neural networks. We envisage that the resolution of FPM will be further enhanced if the Fourier spectrum of the sample outside the synthetic aperture of FPM is accurately predicted.

The integration of neural network and physical reconstruction model for Fourier ptychographic microscopy