Sparsity-regularized coded ptychography for robust and efficient lensless microscopy on a chip

In ptychographic imaging, the trade-off between the number of acquisitions and the resultant imaging quality presents a complex optimization problem. Increasing the number of acquisitions typically yields reconstructions with higher spatial resolution and finer details. Conversely, a reduction in measurement frequency often compromises the quality of the reconstructed images, manifesting as increased noise and coarser details. To address this challenge, we employ sparsity priors to reformulate the ptychographic reconstruction task as a total variation regularized optimization problem. We introduce a new computational framework, termed the ptychographic proximal total-variation (PPTV) solver, designed to integrate into existing ptychography settings without necessitating hardware modifications. Through comprehensive numerical simulations, we validate that PPTV-driven coded ptychography is capable of producing highly accurate reconstructions with a minimal set of eight intensity measurements. Convergence analysis further substantiates the robustness, stability, and computational feasibility of the proposed PPTV algorithm. Experimental results obtained from optical setups unequivocally demonstrate that the PPTV algorithm facilitates high-throughput, high-resolution imaging while significantly reducing the measurement burden. These findings indicate that the PPTV algorithm has the potential to substantially mitigate the resource-intensive requirements traditionally associated with high-quality ptychographic imaging, thereby offering a pathway toward the development of more compact and efficient ptychographic microscopy systems.

Digital staining in optical microscopy using deep learning -- a review

Until recently, conventional biochemical staining had the undisputed status as well-established benchmark for most biomedical problems related to clinical diagnostics, fundamental research and biotechnology. Despite this role as gold-standard, staining protocols face several challenges, such as a need for extensive, manual processing of samples, substantial time delays, altered tissue homeostasis, limited choice of contrast agents for a given sample, 2D imaging instead of 3D tomography and many more. Label-free optical technologies, on the other hand, do not rely on exogenous and artificial markers, by exploiting intrinsic optical contrast mechanisms, where the specificity is typically less obvious to the human observer. Over the past few years, digital staining has emerged as a promising concept to use modern deep learning for the translation from optical contrast to established biochemical contrast of actual stainings. In this review article, we provide an in-depth analysis of the current state-of-the-art in this field, suggest methods of good practice, identify pitfalls and challenges and postulate promising advances towards potential future implementations and applications.

Ptychographic sensor for large-scale lensless microbial monitoring with high spatiotemporal resolution

Traditional microbial detection methods often rely on the overall property of microbial cultures and cannot resolve individual growth event at high spatiotemporal resolution. As a result, they require bacteria to grow to confluence and then interpret the results. Here, we demonstrate the application of an integrated ptychographic sensor for lensless cytometric analysis of microbial cultures over a large scale and with high spatiotemporal resolution. The reported device can be placed within a regular incubator or used as a standalone incubating unit for long-term microbial monitoring. For longitudinal study where massive data are acquired at sequential time points, we report a new temporal-similarity constraint to increase the temporal resolution of ptychographic reconstruction by 7-fold. With this strategy, the reported device achieves a centimeter-scale field of view, a half-pitch spatial resolution of 488 nm, and a temporal resolution of 15-second intervals. For the first time, we report the direct observation of bacterial growth in a 15-second interval by tracking the phase wraps of the recovered images, with high phase sensitivity like that in interferometric measurements. We also characterize cell growth via longitudinal dry mass measurement and perform rapid bacterial detection at low concentrations. For drug-screening application, we demonstrate proof-of-concept antibiotic susceptibility testing and perform single-cell analysis of antibiotic-induced filamentation. The combination of high phase sensitivity, high spatiotemporal resolution, and large field of view is unique among existing microscopy techniques. As a quantitative and miniaturized platform, it can improve studies with microorganisms and other biospecimens at resource-limited settings.

High-throughput lensless whole slide imaging via continuous height-varying modulation of tilted sensor

We report a new lensless microscopy configuration by integrating the concepts of transverse translational ptychography and defocus multi-height phase retrieval. In this approach, we place a tilted image sensor under the specimen for linearly-increasing phase modulation along one lateral direction. Similar to the operation of ptychography, we laterally translate the specimen and acquire the diffraction images for reconstruction. Since the axial distance between the specimen and the sensor varies at different lateral positions, laterally translating the specimen effectively introduces defocus multi-height measurements while eliminating axial scanning. Lateral translation further introduces sub-pixel shift for pixel super-resolution imaging and naturally expands the field of view for rapid whole slide imaging. We show that the equivalent height variation can be precisely estimated from the lateral shift of the specimen, thereby addressing the challenge of precise axial positioning in conventional multi-height phase retrieval. Using a sensor with a 1.67-micron pixel size, our low-cost and field-portable prototype can resolve 690-nm linewidth on the resolution target. We show that a whole slide image of a blood smear with a 120-mm^2 field of view can be acquired in 18 seconds. We also demonstrate accurate automatic white blood cell counting from the recovered image. The reported approach may provide a turnkey solution for addressing point-of-care- and telemedicine-related challenges.

Plug-and-play optimization for pixel super-resolution phase retrieval

In order to increase signal-to-noise ratio in measurement, most imaging detectors sacrifice resolution to increase pixel size in confined area. Although the pixel super-resolution technique (PSR) enables resolution enhancement in such as digital holographic imaging, it suffers from unsatisfied reconstruction quality. In this work, we report a high-fidelity plug-and-play optimization method for PSR phase retrieval, termed as PNP-PSR. It decomposes PSR reconstruction into independent sub-problems based on the generalized alternating projection framework. An alternating projection operator and an enhancing neural network are derived to tackle the measurement fidelity and statistical prior regularization, respectively. In this way, PNP-PSR incorporates the advantages of individual operators, achieving both high efficiency and noise robustness. We compare PNP-PSR with the existing PSR phase retrieval algorithms with a series of simulations and experiments, and PNP-PSR outperforms the existing algorithms with as much as 11dB on PSNR. The enhanced imaging fidelity enables one-order-of-magnitude higher cell counting precision.

Blind deblurring for microscopic pathology images using deep learning networks

Artificial Intelligence (AI)-powered pathology is a revolutionary step in the world of digital pathology and shows great promise to increase both diagnosis accuracy and efficiency. However, defocus and motion blur can obscure tissue or cell characteristics hence compromising AI algorithms'accuracy and robustness in analyzing the images. In this paper, we demonstrate a deep-learning-based approach that can alleviate the defocus and motion blur of a microscopic image and output a sharper and cleaner image with retrieved fine details without prior knowledge of the blur type, blur extent and pathological stain. In this approach, a deep learning classifier is first trained to identify the image blur type. Then, two encoder-decoder networks are trained and used alone or in combination to deblur the input image. It is an end-to-end approach and introduces no corrugated artifacts as traditional blind deconvolution methods do. We test our approach on different types of pathology specimens and demonstrate great performance on image blur correction and the subsequent improvement on the diagnosis outcome of AI algorithms.

Axially-shifted pattern illumination for macroscale turbidity suppression and virtual volumetric confocal imaging without axial scanning

Structured illumination has been widely used for optical sectioning and 3D surface recovery. In a typical implementation, multiple images under non-uniform pattern illumination are used to recover a single object section. Axial scanning of the sample or the objective lens is needed for acquiring the 3D volumetric data. Here we demonstrate the use of axially-shifted pattern illumination (asPI) for virtual volumetric confocal imaging without axial scanning. In the reported approach, we project illumination patterns at a tilted angle with respect to the detection optics. As such, the illumination patterns shift laterally at different z sections and the sample information at different z-sections can be recovered based on the captured 2D images. We demonstrate the reported approach for virtual confocal imaging through a diffusing layer and underwater 3D imaging through diluted milk. We show that we can acquire the entire confocal volume in ~1s with a throughput of 420 megapixels per second. Our approach may provide new insights for developing confocal light ranging and detection systems in degraded visual environments.

Solving Fourier ptychographic imaging problems via neural network modeling and TensorFlow

Fourier ptychography is a recently developed imaging approach for large field-of-view and high-resolution microscopy. Here we model the Fourier ptychographic forward imaging process using a convolution neural network (CNN) and recover the complex object information in the network training process. In this approach, the input of the network is the point spread function in the spatial domain or the coherent transfer function in the Fourier domain. The object is treated as 2D learnable weights of a convolution or a multiplication layer. The output of the network is modeled as the loss function we aim to minimize. The batch size of the network corresponds to the number of captured low-resolution images in one forward / backward pass. We use a popular open-source machine learning library, TensorFlow, for setting up the network and conducting the optimization process. We analyze the performance of different learning rates, different solvers, and different batch sizes. It is shown that a large batch size with the Adam optimizer achieves the best performance in general. To accelerate the phase retrieval process, we also discuss a strategy to implement Fourier-magnitude projection using a multiplication neural network model. Since convolution and multiplication are the two most-common operations in imaging modeling, the reported approach may provide a new perspective to examine many coherent and incoherent systems. As a demonstration, we discuss the extensions of the reported networks for modeling single-pixel imaging and structured illumination microscopy (SIM). 4-frame resolution doubling is demonstrated using a neural network for SIM. We have made our implementation code open-source for the broad research community.

Rapid focus map surveying for whole slide imaging with continues sample motion

Whole slide imaging (WSI) has recently been cleared for primary diagnosis in the US. A critical challenge of WSI is to perform accurate focusing in high speed. Traditional systems create a focus map prior to scanning. For each focus point on the map, sample needs to be static in the x-y plane and axial scanning is needed to maximize the contrast. Here we report a novel focus map surveying method for WSI. The reported method requires no axial scanning, no additional camera and lens, works for stained and transparent samples, and allows continuous sample motion in the surveying process. It can be used for both brightfield and fluorescence WSI. By using a 20X, 0.75 NA objective lens, we demonstrate a mean focusing error of ~0.08 microns in the static mode and ~0.17 microns in the continuous motion mode. The reported method may provide a turnkey solution for most existing WSI systems for its simplicity, robustness, accuracy, and high-speed. It may also standardize the imaging performance of WSI systems for digital pathology and find other applications in high-content microscopy such as DNA sequencing and time-lapse live-cell imaging.