Fly-scan high-throughput coded ptychographic microscopy via active micro-vibration and rolling-shutter distortion correction.

Recent advancements in ptychography have demonstrated the potential of coded ptychography (CP) for high-resolution optical imaging in a lensless configuration. However, CP suffers imaging throughput limitations due to scanning inefficiencies. To address this, we propose what we believe is a novel 'fly-scan' scanning strategy utilizing two eccentric rotating mass (ERM) vibration motors for high-throughput coded ptychographic microscopy. The intrinsic continuity of the 'fly-scan' technique effectively eliminates the scanning overhead typically encountered during data acquisition. Additionally, its randomized scanning trajectory considerably reduces periodic artifacts in image reconstruction. We also developed what we believe to be a novel rolling-shutter distortion correction algorithm to fix the rolling-shutter effects. We built up a low-cost, DIY-made prototype platform and validated our approach with various samples including a resolution target, a quantitative phase target, a thick potato sample and biospecimens. The reported platform may offer a cost-effective and turnkey solution for high-throughput bio-imaging.

Structured modulation multi-height microscopy for high-resolution imaging.

Conventional multi-height microscopy techniques introduce different object-to-detector distances to obtain multiple measurements for phase retrieval. However, surpassing the diffraction limit imposed by the numerical aperture (NA) of the objective lens remains a challenging task. Here, we report a novel structured modulation multi-height microscopy technique for quantitative high-resolution imaging. In our platform, a thin diffuser is placed in between the sample and the objective lens. By translating the diffuser to different axial positions, a sequence of modulated intensity images is captured for reconstruction. The otherwise inaccessible high-resolution object information can thus be encoded into the optical system for detection. In the construction process, we report a ptychographic phase retrieval algorithm to recover the existing wavefront of the complex object. We validate our approach using a resolution target, a phase target, and various biological samples. We demonstrate a ∼4-fold resolution gain over the diffraction limit. We also demonstrate our approach to achieve a 6.5 mm by 4.3 mm field of view and a half-pitch resolution of 1.2 µm. The reported methodology provides a portable, turnkey solution for quantitative high-resolution imaging with potential applications in disease diagnosis, sample screening, and other fields.

Remote referencing strategy for high-resolution coded ptychographic imaging.

The applications of conventional ptychography are limited by its relatively low resolution and throughput in the visible light regime. The new development of coded ptychography (CP) has addressed these issues and achieved the highest numerical aperture for large-area optical imaging in a lensless configuration. A high-quality reconstruction of CP relies on precise tracking of the coded sensor's positional shifts. The coded layer on the sensor, however, prevents the use of cross correlation analysis for motion tracking. Here we derive and analyze the motion tracking model of CP. A novel, to the best of our knowledge, remote referencing scheme and its subsequent refinement pipeline are developed for blind image acquisition. By using this approach, we can suppress the correlation peak caused by the coded surface and recover the positional shifts with deep sub-pixel accuracy. In contrast with common positional refinement methods, the reported approach can be disentangled from the iterative phase retrieval process and is computationally efficient. It allows blind image acquisition without motion feedback from the scanning process. It also provides a robust and reliable solution for implementing ptychography with high imaging throughput. We validate this approach by performing high-resolution whole slide imaging of bio-specimens.

Ptychography-based high-throughput lensless on-chip microscopy via incremental proximal algorithms.

Ptychography-based lensless on-chip microscopy enables high-throughput imaging by retrieving the missing phase information from intensity measurements. Numerous reconstruction algorithms for ptychography have been proposed, yet only a few incremental algorithms can be extended to lensless on-chip microscopy because of large-scale datasets but limited computational efficiency. In this paper, we propose the use of accelerated proximal gradient methods for blind ptychographic phase retrieval in lensless on-chip microscopy. Incremental gradient approaches are adopted in the reconstruction routine. Our algorithms divide the phase retrieval problem into sub-problems involving the evaluation of proximal operator, stochastic gradient descent, and Wirtinger derivatives. We benchmark the performances of accelerated proximal gradient, extended ptychographic iterative engine, and alternating direction method of multipliers, and discuss their convergence and accuracy in both noisy and noiseless cases. We also validate our algorithms using experimental datasets, where full field of view measurements are captured to recover the high-resolution complex samples. Among these algorithms, accelerated proximal gradient presents the overall best performance regarding accuracy and convergence rate. The proposed methods may find applications in ptychographic reconstruction, especially for cases where a wide field of view and high resolution are desired at the same time.

Deep learning-enabled whole slide imaging (DeepWSI): oil-immersion quality using dry objectives, longer depth of field, higher system throughput, and better functionality.

Whole slide imaging (WSI) has moved the traditional manual slide inspection process to the era of digital pathology. A typical WSI system translates the sample to different positions and captures images using a high numerical aperture (NA) objective lens. Performing oil-immersion microscopy is a major obstacle for WSI as it requires careful liquid handling during the scanning process. Switching between dry objective and oil-immersion lens is often impossible as it disrupts the acquisition process. For a high-NA objective lens, the sub-micron depth of field also poses a challenge to acquiring in-focus images of samples with uneven topography. Additionally, it implies a small field of view for each tile, thus limiting the system throughput and resulting in a long acquisition time. Here we report a deep learning-enabled WSI platform, termed DeepWSI, to substantially improve the system performance and imaging throughput. With this platform, we show that images captured with a regular dry objective lens can be transformed into images comparable to that of a 1.4-NA oil immersion lens. Blurred images with defocus distance from -5 µm to +5 µm can be virtually refocused to the in-focus plane post measurement. We demonstrate an equivalent data throughput of >2 gigapixels per second, the highest among existing WSI systems. Using the same deep neural network, we also report a high-resolution virtual staining strategy and demonstrate it for Fourier ptychographic WSI. The DeepWSI platform may provide a turnkey solution for developing high-performance diagnostic tools for digital pathology.

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

We report a new, to the best of our knowledge, 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 introducing 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 1.67 µm pixel size, our low-cost and field-portable prototype can resolve the 690 nm linewidth on the resolution target. We show that a whole slide image of a blood smear with a 120mm2 field of view can be acquired in 18 s. 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.

Virtual brightfield and fluorescence staining for Fourier ptychography via unsupervised deep learning.

Fourier ptychographic microscopy (FPM) is a computational approach geared towards creating high-resolution and large field-of-view images without mechanical scanning. Acquiring color images of histology slides often requires sequential acquisitions with red, green, and blue illuminations. The color reconstructions often suffer from coherent artifacts that are not presented in regular incoherent microscopy images. As a result, it remains a challenge to employ FPM for digital pathology applications, where resolution and color accuracy are of critical importance. Here we report a deep learning approach for performing unsupervised image-to-image translation of FPM reconstructions. A cycle-consistent adversarial network with multiscale structure similarity loss is trained to perform virtual brightfield and fluorescence staining of the recovered FPM images. In the training stage, we feed the network with two sets of unpaired images: (1) monochromatic FPM recovery and (2) color or fluorescence images captured using a regular microscope. In the inference stage, the network takes the FPM input and outputs a virtually stained image with reduced coherent artifacts and improved image quality. We test the approach on various samples with different staining protocols. High-quality color and fluorescence reconstructions validate its effectiveness.

Super-resolved multispectral lensless microscopy via angle-tilted, wavelength-multiplexed ptychographic modulation.

We report an angle-tilted, wavelength-multiplexed ptychographic modulation approach for multispectral lensless on-chip microscopy. In this approach, we illuminate the specimen with lights at five wavelengths simultaneously. A prism is added at the illumination path for spectral dispersion. Thus, lightwaves at different wavelengths hit the specimen at slightly different incident angles, breaking the ambiguities in mixed-state ptychographic reconstruction. At the detection path, we place a thin diffuser between the specimen and the monochromatic image sensor for encoding the spectral information into 2D intensity measurements. By scanning the sample to different x-y positions, we acquire a sequence of monochromatic images for reconstructing the five complex object profiles at the five wavelengths. An up-sampling procedure is integrated into the recovery process to bypass the resolution limit imposed by the imager pixel size. We demonstrate a half-pitch resolution of 0.55 µm using an image sensor with 1.85 µm pixel size. We also demonstrate quantitative and high-quality multispectral reconstructions of stained tissue sections for digital pathology applications.

Super-resolution microscopy via ptychographic structured modulation of a diffuser.

We report a new coherent imaging technique, termed ptychographic structured modulation (PSM), for quantitative super-resolution microscopy. In this technique, we place a thin diffuser (i.e., a scattering lens) in between the sample and the objective lens to modulate the complex light waves from the object. The otherwise inaccessible high-resolution object information can thus be encoded into the captured images. We then employ a ptychographic phase retrieval process to jointly recover the exit wavefront of the complex object and the unknown diffuser profile. Unlike the illumination-based super-resolution approach, the recovered image of our approach depends upon how the complex wavefront exits the sample-not enters it. Therefore, the sample thickness becomes irrelevant during reconstruction. After recovery, we can propagate the super-resolution complex wavefront to any position along the optical axis. We validate our approach using a resolution target, a quantitative phase target, a two-layer sample, and a thick polydimethylsiloxane sample. We demonstrate a 4.5-fold resolution gain over the diffraction limit. We also show that a four-fold resolution gain can be achieved with as few as ∼30 images. The reported approach may provide a quantitative super-resolution strategy for coherent light, x-ray, and electron imaging.

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 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 3D sample information 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 ∼1 s 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.

Field-portable quantitative lensless microscopy based on translated speckle illumination and sub-sampled ptychographic phase retrieval.

We report a compact, cost-effective, and field-portable lensless imaging platform for quantitative microscopy. In this platform, the object is placed on top of an image sensor chip without using a lens. We use a low-cost galvo scanner to rapidly scan an unknown laser speckle pattern on the object. To address the positioning repeatability and accuracy issues, we directly recover the positional shifts of the speckle pattern based on the phase correlation of the captured images. To bypass the resolution limit set by the imager pixel size, we employ a sub-sampled ptychographic phase retrieval process to recover the complex object. We validate our approach using a resolution target, phase target, and biological sample. Our results show that accurate, high-quality complex images can be obtained from a lensless dataset with as few as ∼10 images. We also demonstrate the reported approach to achieve a 6.4-mm by 4.6-mm field of view and a half-pitch resolution of 1 µm. The reported approach may provide a quantitative lensless imaging strategy for addressing point-of-care-, global-health-, and telemedicine-related challenges.

Fourier ptychographic microscopy using a generalized Anscombe transform approximation of the mixed Poisson-Gaussian likelihood.

Fourier ptychographic microscopy (FPM) is a novel computational microscopy technique that provides intensity images with both wide field-of-view (FOV) and high-resolution (HR). By combining ideas from synthetic aperture and phase retrieval, FPM iteratively stitches together a number of variably illuminated, low-resolution (LR) intensity images in Fourier space to reconstruct an HR complex sample image. In practice, however, the reconstruction of FPM is sensitive to the input noise, including Gaussian noise, Poisson shot noise or mixed Poisson-Gaussian noise. To efficiently address these noises, we developed a novel FPM reconstruction method termed generalized Anscombe transform approximation Fourier ptychographic (GATFP) reconstruction. The method utilizes the generalized Anscombe transform (GAT) approximation for the noise model, and a maximum likelihood theory is employed for formulating the FPM optimization problem. We validated the proposed method with both simulated data for quantitative evaluation and real experimental data captured using FPM setup. The results show that the proposed method achieves state-of-the-art performance in comparison with other approaches.

Ptycho-endoscopy on a lensless ultrathin fiber bundle tip.

Synthetic aperture radar (SAR) utilizes an aircraft-carried antenna to emit electromagnetic pulses and detect the returning echoes. As the aircraft travels across a designated area, it synthesizes a large virtual aperture to improve image resolution. Inspired by SAR, we introduce synthetic aperture ptycho-endoscopy (SAPE) for micro-endoscopic imaging beyond the diffraction limit. SAPE operates by hand-holding a lensless fiber bundle tip to record coherent diffraction patterns from specimens. The fiber cores at the distal tip modulate the diffracted wavefield within a confined area, emulating the role of the 'airborne antenna' in SAR. The handheld operation introduces positional shifts to the tip, analogous to the aircraft's movement. These shifts facilitate the acquisition of a ptychogram and synthesize a large virtual aperture extending beyond the bundle's physical limit. We mitigate the influences of hand motion and fiber bending through a low-rank spatiotemporal decomposition of the bundle's modulation profile. Our tests demonstrate the ability to resolve a 548-nm linewidth on a resolution target. The achieved space-bandwidth product is ~1.1 million effective pixels, representing a 36-fold increase compared to that of the original fiber bundle. Furthermore, SAPE's refocusing capability enables imaging over an extended depth of field exceeding 2 cm. The aperture synthesizing process in SAPE surpasses the diffraction limit set by the probe's maximum collection angle, opening new opportunities for both fiber-based and distal-chip endoscopy in applications such as medical diagnostics and industrial inspection.

Optical ptychography for biomedical imaging: recent progress and future directions [Invited].

Ptychography is an enabling microscopy technique for both fundamental and applied sciences. In the past decade, it has become an indispensable imaging tool in most X-ray synchrotrons and national laboratories worldwide. However, ptychography's limited resolution and throughput in the visible light regime have prevented its wide adoption in biomedical research. Recent developments in this technique have resolved these issues and offer turnkey solutions for high-throughput optical imaging with minimum hardware modifications. The demonstrated imaging throughput is now greater than that of a high-end whole slide scanner. In this review, we discuss the basic principle of ptychography and summarize the main milestones of its development. Different ptychographic implementations are categorized into four groups based on their lensless/lens-based configurations and coded-illumination/coded-detection operations. We also highlight the related biomedical applications, including digital pathology, drug screening, urinalysis, blood analysis, cytometric analysis, rare cell screening, cell culture monitoring, cell and tissue imaging in 2D and 3D, polarimetric analysis, among others. Ptychography for high-throughput optical imaging, currently in its early stages, will continue to improve in performance and expand in its applications. We conclude this review article by pointing out several directions for its future development.

Depth-multiplexed ptychographic microscopy for high-throughput imaging of stacked bio-specimens on a chip.

Imaging a large number of bio-specimens at high speed is essential for many biomedical applications. The common strategy is to place specimens at different lateral positions and image them sequentially. Here we report a new on-chip imaging strategy, termed depth-multiplexed ptychographic microscopy (DPM), for parallel imaging and sensing at high speed. Different from the common strategy, DPM stacks multiple specimens in the axial direction and images the entire z-stack all at once. In our prototype platform, we modify a low-cost car mirror for programmable steering of the incident laser beam. A blood-coated image sensor is then placed underneath the stacked sample for acquiring the resulting diffraction patterns. With the captured images, we perform blind recovery of the incident beam angle and model different layers of the stacked sample as different coded surfaces for object reconstruction. For in vitro experiment, we demonstrate time-lapse cell culture monitoring by imaging 3 stacked microfluidic channels on the coded sensor. For high-throughput cytometric analysis, we image 5 stacked brain sections with a 205-mm2 field of view in ∼50 s. Cytometric analysis is also performed to quantify the cellular proliferation biomarkers on the slides. The DPM approach adds a new degree of freedom for data multiplexing in microscopy, enabling parallel imaging of multiple specimens using a single detector. The demonstrated 6-mm depth of field is among the longest ones in microscopy imaging. The novel depth-multiplexed configuration also complements the miniaturization provided by microfluidics devices, offering a solution for on-chip sensing and imaging with efficient sample handling.

Spatial- and Fourier-domain ptychography for high-throughput bio-imaging.

First envisioned for determining crystalline structures, ptychography has become a useful imaging tool for microscopists. However, ptychography remains underused by biomedical researchers due to its limited resolution and throughput in the visible light regime. Recent developments of spatial- and Fourier-domain ptychography have successfully addressed these issues and now offer the potential for high-resolution, high-throughput optical imaging with minimal hardware modifications to existing microscopy setups, often providing an excellent trade-off between resolution and field of view inherent to conventional imaging systems, giving biomedical researchers the best of both worlds. Here, we provide extensive information to enable the implementation of ptychography by biomedical researchers in the visible light regime. We first discuss the intrinsic connections between spatial-domain coded ptychography and Fourier ptychography. A step-by-step guide then provides the user instructions for developing both systems with practical examples. In the spatial-domain implementation, we explain how a large-scale, high-performance blood-cell lens can be made at negligible expense. In the Fourier-domain implementation, we explain how adding a low-cost light source to a regular microscope can improve the resolution beyond the limit of the objective lens. The turnkey operation of these setups is suitable for use by professional research laboratories, as well as citizen scientists. Users with basic experience in optics and programming can build the setups within a week. The do-it-yourself nature of the setups also allows these procedures to be implemented in laboratory courses related to Fourier optics, biomedical instrumentation, digital image processing, robotics and capstone projects.

High-throughput digital pathology via a handheld, multiplexed, and AI-powered ptychographic whole slide scanner.

The recent advent of whole slide imaging (WSI) systems has moved digital pathology closer to diagnostic applications and clinical practices. Integrating WSI with machine learning promises the growth of this field in upcoming years. Here we report the design and implementation of a handheld, colour-multiplexed, and AI-powered ptychographic whole slide scanner for digital pathology applications. This handheld scanner is built using low-cost and off-the-shelf components, including red, green, and blue laser diodes for sample illumination, a modified stage for programmable sample positioning, and a synchronized image sensor pair for data acquisition. We smear a monolayer of goat blood cells on the main sensor for high-resolution lensless coded ptychographic imaging. The synchronized secondary sensor acts as a non-contact encoder for precisely tracking the absolute object position for ptychographic reconstruction. For WSI, we introduce a new phase-contrast-based focus metric for post-acquisition autofocusing of both stained and unstained specimens. We show that the scanner can resolve the 388-nm linewidth on the resolution target and acquire gigapixel images with a 14 mm × 11 mm area in ∼70 seconds. The imaging performance is validated with regular stained pathology slides, unstained thyroid smears, and malaria-infected blood smears. The deep neural network developed in this study further enables high-throughput cytometric analysis using the recovered complex amplitude. The reported do-it-yourself scanner offers a portable solution to transform the high-end WSI system into one that can be made widely available at a low cost. The capability of high-throughput quantitative phase imaging may also find applications in rapid on-site evaluations.

Blood-Coated Sensor for High-Throughput Ptychographic Cytometry on a Blu-ray Disc.

The Blu-ray drive is an engineering masterpiece that integrates disc rotation, pickup head translation, and three lasers in a compact and portable format. Here, we integrate a blood-coated image sensor with a modified Blu-ray drive for high-throughput cytometric analysis of various biospecimens. In this device, samples are mounted on the rotating Blu-ray disc and illuminated by the built-in lasers from the pickup head. The resulting coherent diffraction patterns are then recorded by the blood-coated image sensor. The rich spatial features of the blood-cell monolayer help down-modulate the object information for sensor detection, thus forming a high-resolution computational biolens with a theoretically unlimited field of view. With the acquired data, we develop a lensless coherent diffraction imaging modality termed rotational ptychography for image reconstruction. We show that our device can resolve the 435 nm line width on the resolution target and has a field of view only limited by the size of the Blu-ray disc. To demonstrate its applications, we perform high-throughput urinalysis by locating disease-related calcium oxalate crystals over the entire microscope slide. We also quantify different types of cells on a blood smear with an acquisition speed of ∼10,000 cells per second. For in vitro experiments, we monitor live bacterial cultures over the entire Petri dish with single-cell resolution. Using biological cells as a computational lens could enable new intriguing imaging devices for point-of-care diagnostics. Modifying a Blu-ray drive with the blood-coated sensor further allows the spread of high-throughput optical microscopy from well-equipped laboratories to citizen scientists worldwide.

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-s intervals. For the first time, we report the direct observation of bacterial growth in a 15-s 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.

Quantitative multi-height phase retrieval via a coded image sensor.

Multi-height phase retrieval introduces different object-to-detector distances for obtaining phase diversity measurements. In the acquisition process, the slow-varying phase information, however, cannot be converted to intensity variations for detection. Therefore, the low-frequency contents of the phase profile are lost during acquisition and cannot be properly restored via phase retrieval. Here, we demonstrate the use of a coded image sensor for addressing this challenge in multi-height phase retrieval. In our scheme, we add a coded layer on top of the image sensor for encoding the slow-varying complex wavefronts into intensity variations of the modulated patterns. Inspired by the concept of blind ptychography, we report a reconstruction scheme to jointly recover the complex object and the unknown coded layer using multi-height measurements. With both simulation and experimental results, we show that the recovered phase is quantitative and the slow-varying phase profiles can be properly restored using lensless multi-height measurements. We also show that the image quality using the coded sensor is better than that of a regular image sensor. For demonstrations, we validate the reported scheme with various biospecimens and compare the results to those of regular lensless multi-height phase retrieval. The use of a coded image sensor may enable true quantitative phase imaging for the lensless multi-height, multi-wavelength, and transport-of-intensity equation approaches.