We discuss a coded ptychography technique that significantly enhances imaging resolution and throughput, achieving an order of magnitude improvement over previous methods. Our platform involves translating samples across disorder-engineered surfaces for lensless diffraction data acquisition. These engineered surfaces can be created by smearing a monolayer of blood on the image sensor. By monitoring the phase wraps of the recovered images, we have successfully observed bacterial growth at 15-second intervals over a 120 mm^2 area, achieving phase sensitivity comparable to interferometric measurements. For drug-screening applications, we demonstrate proof-of-concept rapid antibiotic susceptibility testing. By adopting a depth-multiplexed configuration, our approach can be extended to simultaneously image a stack of biospecimens. The fusion of high phase sensitivity, superior spatiotemporal resolution, an ultra-large field of view, and depth-multiplexing capability sets the coded ptychographic microscopy apart from existing imaging techniques, marking an important advancement for high-throughput label-free lensless imaging.
KEYWORDS: Neural networks, Wavefronts, Coherence imaging, Biological imaging, Data modeling, Holography, Convolution, Super resolution, Image restoration, Education and training
Large-scale computational imaging can provide remarkable space-bandwidth product that is beyond the limit of optical systems. In coherent imaging (CI), the joint reconstruction of amplitude and phase further expands the information throughput and sheds light on label-free observation of biological samples at micro- or even nano-levels. The existing large-scale CI techniques usually require scanning/modulation multiple times to guarantee measurement diversity and long exposure time to achieve a high signal-to-noise ratio. Such cumbersome procedures restrict clinical applications for rapid and low-phototoxicity cell imaging. In this work, a complex-domain-enhancing neural network for large-scale CI termed CI-CDNet is proposed for various large-scale CI modalities with satisfactory reconstruction quality and efficiency. CI-CDNet is able to exploit the latent coupling information between amplitude and phase (such as their same features), realizing multidimensional representations of the complex wavefront. The cross-field characterization framework empowers strong generalization and robustness for various coherent modalities, allowing high-quality and efficient imaging under extremely low exposure time and few data volume. We apply CI-CDNet in various large-scale CI modalities including Kramers–Kronig-relations holography, Fourier ptychographic microscopy, and lensless coded ptychography. A series of simulations and experiments validate that CI-CDNet can reduce exposure time and data volume by more than 1 order of magnitude. We further demonstrate that the high-quality reconstruction of CI-CDNet benefits the subsequent high-level semantic analysis.
Optical imaging has served as a primary method to collect information about biosystems across scales—from functionalities of tissues to morphological structures of cells and even at biomolecular levels. However, to adequately characterize a complex biosystem, an imaging system with a number of resolvable points, referred to as a space-bandwidth product (SBP), in excess of one billion is typically needed. Since a gigapixel-scale far exceeds the capacity of current optical imagers, compromises must be made to obtain either a low spatial resolution or a narrow field-of-view (FOV). The problem originates from constituent refractive optics—the larger the aperture, the more challenging the correction of lens aberrations. Therefore, it is impractical for a conventional optical imaging system to achieve an SBP over hundreds of millions. To address this unmet need, a variety of high-SBP imagers have emerged over the past decade, enabling an unprecedented resolution and FOV beyond the limit of conventional optics. We provide a comprehensive survey of high-SBP imaging techniques, exploring their underlying principles and applications in bioimaging.
We discuss two compact, cost-effective, and field-portable ptychographic lensless imaging platforms for quantitative microscopy. In the first implementation, 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. In the second implementation, we place a thin diffuser in between the object and the image sensor for light wave modulation. By blindly scanning the unknown diffuser to different x-y positions, we acquire a sequence of modulated intensity images for quantitative object recovery. Different from previous ptychographic implementations, we employ a unit magnification configuration with a Fresnel number of ~50,000, which is orders of magnitude higher than previous ptychographic setups. The unit magnification configuration allows us to have the entire sensor area, 6.4 mm by 4.6 mm, as the imaging field of view. The ultra-high Fresnel number enables us to directly recover the positional shift of the diffuser in the phase retrieval process. In this second implementation, we use a low-cost, DIY scanning stage to perform blind diffuser modulation. We further employ an up-sampling phase retrieval scheme to bypass the resolution limit set by the imager pixel size and demonstrate a half-pitch resolution of 0.78 µm. For both implementations, we validate the imaging performance via various biological samples. The reported platforms provide cost-effective and turnkey solutions for large field-of-view, high-resolution, and quantitative on-chip microscopy. They are adaptable for a wide range of point-of-care-, global-health-, and telemedicine-related applications.
Digital pathology via whole-slide imaging (WSI) systems has recently been approved for the primary diagnostic use in the US. Acquiring whole-slide images with spectral information at each pixel permits the use of multiplexed antibody labeling and allow for the measurement of cellularly resolved chemical information. Here, we report the development of a high-throughput terapixel hyperspectral WSI system using prism-based slit-array dispersion. We demonstrate a slit-array detection scheme for absorption-based measurements and a slit-array projection scheme for fluorescence-based measurements. The spectral resolution and spectral range in the reported schemes can be adjusted by changing the orientation of the slit-array mask. We use our system to acquire 74 5-megapixel brightfield images at different wavelengths in ∼1 s, corresponding to a throughput of 0.375 gigapixels / s. A terapixel whole-slide spatial–spectral data cube can be obtained in ∼45 min. The reported system is compatible with existing WSI systems and can be developed as an add-on module for whole-slide spectral imaging. It may find broad applications in high-throughput chemical imaging with multiple antibody labeling. The use of slit array for structured illumination may also provide insights for developing high-throughput hyperspectral confocal imaging systems.
Fourier ptychographic microscopy (FPM) is a recently developed technique stitching low-resolution images in Fourier domain to realize wide-field high-resolution imaging. However, the time-consuming process of image acquisition greatly narrows its applications in dynamic imaging. We report a wavelength multiplexing strategy to speed up the acquisition process of FPM several folds. A proof-of-concept system is built to verify its feasibility. Distinguished from many current multiplexing methods in Fourier domain, we explore the potential of high-speed FPM in spectral domain. Compatible with most existing FPM methods, our strategy provides an approach to high-speed gigapixel microscopy. Several experimental results are also presented to validate the strategy.
Scanning confocal microscopy is a standard choice for many fluorescence imaging applications in basic biomedical research. It is able to produce optically sectioned images and provide acquisition versatility to address many samples and application demands. However, scanning a focused point across the specimen limits the speed of image acquisition. As a result, scanning confocal microscope only works well with stationary samples. Researchers have performed parallel confocal scanning using digital-micromirror-device (DMD), which was used to project a scanning multi-point pattern across the sample. The DMD based parallel confocal systems increase the imaging speed while maintaining the optical sectioning ability. In this paper, we report the development of an add-on kit for high-speed and low-cost confocal microscopy. By adapting this add-on kit to an existing regular microscope, one can convert it into a confocal microscope without significant hardware modifications. Compared with current DMD-based implementations, the reported approach is able to recover multiple layers along the z axis simultaneously. It may find applications in wafer inspection and 3D metrology of semiconductor circuit. The dissemination of the proposed add-on kit under $1000 budget could also lead to new types of experimental designs for biological research labs, e.g., cytology analysis in cell culture experiments, genetic studies on multicellular organisms, pharmaceutical drug profiling, RNA interference studies, investigation of microbial communities in environmental systems, and etc.
The Fourier ptychography technique in reflection mode has great potential applications in tissue imaging and optical inspection, but the current configuration either has a limitation on cut-off frequency or is not practical. By placing the imaging aperture stop outside the illumination path, the illumination numerical aperture (NA) can be greater than the imaging NA of the objective lens. Thus, the cut-off frequency achieved in the proposed optical system is greater than twice the objective’s NA divided by the wavelength (2NAobj/λ), which is the diffraction limit for the cut-off frequency in an incoherent epi-illumination configuration. We experimentally demonstrated that the synthesized NA is increased by a factor of 4.5 using the proposed optical concept. The key advantage of the proposed system is that it can achieve high-resolution imaging over a large field of view with a simple objective. It will have a great potential for applications in endoscopy, biomedical imaging, surface metrology, and industrial inspection.
Circulating tumor cells (CTCs) are recognized as a candidate biomarker with strong prognostic and predictive potential in metastatic disease. Filtration-based enrichment technologies have been used for CTC characterization, and our group has previously developed a membrane microfilter device that demonstrates efficacy in model systems and clinical blood samples. However, uneven filtration surfaces make the use of standard microscopic techniques a difficult task, limiting the performance of automated imaging using commercially available technologies. Here, we report the use of Fourier ptychographic microscopy (FPM) to tackle this challenge. Employing this method, we were able to obtain high-resolution color images, including amplitude and phase, of the microfilter samples over large areas. FPM’s ability to perform digital refocusing on complex images is particularly useful in this setting as, in contrast to other imaging platforms, we can focus samples on multiple focal planes within the same frame despite surface unevenness. In model systems, FPM demonstrates high image quality, efficiency, and consistency in detection of tumor cells when comparing corresponding microfilter samples to standard microscopy with high correlation (R2=0.99932). Based on these results, we believe that FPM will have important implications for improved, high throughput, filtration-based CTC analysis, and, more generally, image analysis of uneven surfaces.
The on-chip detection of a weak optical signal in biological experiments can easily be complicated by the presence of an
overwhelming background signal, and as such, pre-detection background suppression is substantively important for
weak signal detection. In this paper, we report a structure that can be directly incorporated onto optical sensors to
accomplish background suppression prior to detection. This structure, termed surface-wave-enabled darkfield aperture
(SWEDA), consists of a central sub-wavelength hole surrounded by concentric grooves that are milled onto a gold layer.
Incoming light can be collected and converted into surface waves (SW) by the concentric grooves and then be recoupled
into propagating light through the central hole. We show that the SW-assisted optical component and the direct
transmission component of the central hole can cancel each other, resulting in near-zero transmission under uniform
illumination (observed suppression factor of 1230). This structure can therefore be used to suppress a light field's bright
background and allow sensitive detection of localized light field non-uniformity (observed image contrast enhancement
of 27dB). We also show that under a coherent background illumination, a CMOS pixel patterned with the proposed
structure achieves better SNR performance than an un-patterned single pixel.
We have developed a novel microscope technique that can achieve wide field-of-view (FOV) imaging and yet possess
resolution that is comparable to conventional microscope. The principle of wide FOV microscope system breaks the link
between resolution and FOV magnitude of traditional microscopes. Furthermore, by eliminating bulky optical elements
from its design and utilizing holographic optical elements, the wide FOV microscope system is more cost-effective. In
our system, a hologram was made to focus incoming collimated beam into a focus grid. The sample is put in the focal
plane and the transmissions of the focuses are detected by an imaging sensor. By scanning the incident angle of the
incoming beam, the focus grid will scan across the sample and the time-varying transmission can be detected. We can
then reconstruct the transmission image of the sample. The resolution of microscopic image is limited by the size of the
focus formed by the hologram. The scanning area of each focus spot is determined by the separation of the focus spots
and can be made small for fast imaging speed. We have fabricated a prototype system with a 2.4-mm FOV and 1-μm
resolution. The prototype system was used to image onion skin cells for a demonstration. The preliminary experiments
prove the feasibility of the wide FOV microscope technique, and the possibility of a wider FOV system with better resolution.
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