Sex differences in zymosan-induced behavioral visceral hypersensitivity and colorectal afferent sensitization.

Sex differences in visceral nociception have been reported in clinical and preclinical studies, but the potential differences in sensory neural encoding of the colorectum between males and females are not well understood. In this study, we systematically assessed sex differences in colorectal neural encoding by conducting high-throughput optical recordings in intact dorsal root ganglia (DRGs) from control and visceral hypersensitive mice. We found an apparent sex difference in zymosan-induced behavioral visceral hypersensitivity: enhanced visceromotor responses to colorectal distension were observed only in male mice, not in female mice. In addition, a higher number of mechanosensitive colorectal afferents were identified per mouse in the zymosan-treated male group than in the saline-treated male group, whereas the mechanosensitive afferents identified per mouse were comparable between the zymosan- and saline-treated female groups. The increased number of identified afferents in zymosan-treated male mice was predominantly from thoracolumbar (TL) innervation, which agrees with the significant increase in the TL afferent proportion in the zymosan group as compared with the control group in male mice. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline- and zymosan-treated groups. Our results revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity, which could drive differential clinical symptoms in male and female patients.NEW & NOTEWORTHY We used high-throughput GCaMP6f recordings to study 2,275 mechanosensitive colorectal afferents in mice. Our results revealed significant sex differences in the zymosan-induced behavioral visceral hypersensitivity, which were present in male but not female mice. Male mice also showed sensitization of colorectal afferents in the thoracolumbar pathway, whereas female mice did not. These findings highlight sex differences in sensory neural anatomy and function of the colorectum, with implications for sex-specific therapies for treating visceral pain.

Unraveling Anisotropic and Pulsating Etching of ZnO Nanorods in Hydrochloric Acid via Correlative Electron Microscopy.

Despite much technical progress achieved so far, the exact surface and shape evolution during wet chemical etching is less unraveled, especially in ionically bonded ceramics. Herein, by using in situ liquid cell transmission electron microscopy, a repeated two-stage anisotropic and pulsating periodic etching dynamic is discovered during the pencil shape evolution of a single crystal ZnO nanorod in aqueous hydrochloric acid. Specifically, the nanopencil tip shrinks at a slower rate along [0001̅] than that along the ⟨101̅0⟩ directions, resulting in a sharper ZnO pencil tip. Afterward, rapid tip dissolution happens due to accelerated etching rates along various crystal directions. Concurrently, the vicinal base region of the original nanopencil tip emerges as a new tip followed by the repeated sequence of tip shrinking and removal. The high-index surfaces, such as {101̅m} (m = 0, 1, 2, or 3) and {21̅ 1̅n} (n = 0, 1, 2, or 3), are found to preferentially expose in different ratios. Our 3D electron tomography, convergent beam electron diffraction, middle-angle bright-field STEM, and XPS results indicate the dissociative Cl- species were bound to the Zn-terminated tip surfaces. Furthermore, DFT calculation suggests the preferential Cl- passivation over the {101̅1} and (0001) surfaces of lower energy than others, leading to preferential surface exposures and the oscillatory variation of different facet etching rates. The boosted reactivity due to high-index nanoscale surface exposures is confirmed by comparatively enhanced chemical sensing and CO2 hydrogenation activity. These findings provide an in-depth understanding of anisotropic wet chemical etching of ionic nanocrystals and offer a design strategy for advanced functional materials.

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.

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.

Brightfield, fluorescence, and phase-contrast whole slide imaging via dual-LED autofocusing.

Whole slide imaging (WSI) systems convert the conventional biological samples into digital images. Existing commercial WSI systems usually require an expensive high-performance motorized stage to implement the precise mechanical control, and the cost is prohibitive for most individual pathologists. In this work, we report a low-cost WSI system using the off-the-shelf components, including a computer numerical control (CNC) router, a photographic lens, a programmable LED array, a fluorescence filter cube, and a surface-mount LED. To perform real-time single-frame autofocusing, we exploited two elements of a programmable LED array to illuminate the sample from two different incident angles. The captured image would contain two copies of the sample with a certain separation determined by the defocus distance of the sample. Then the defocus distance can be recovered by identifying the translational shift of the two copies. The reported WSI system can reach a resolution of ∼0.7 µm. The time to determine the optimal focusing position for each tile is only 0.02 s, which is about an 83% improvement compared to our previous work. We quantified the focusing performance on 1890 different tissue tiles. The mean focusing error is ∼0.34 µm, which is well below the ± 0.7 µm depth of field range of our WSI system. The reported WSI system can handle both the semitransparent and the transparent sample, enabling us to demonstrate the implementation of brightfield, fluorescence, and phase-contrast WSI. An automatic digital distortion correction strategy is also developed to avoid the stitching errors. The reported prototype has an affordable cost and can make it broadly available and utilizable for individual pathologists as well as can promote the development of digital pathology.

High-Throughput Functional Characterization of Visceral Afferents by Optical Recordings From Thoracolumbar and Lumbosacral Dorsal Root Ganglia.

Functional understanding of visceral afferents is important for developing the new treatment to visceral hypersensitivity and pain. The sparse distribution of visceral afferents in dorsal root ganglia (DRGs) has challenged conventional electrophysiological recordings. Alternatively, Ca2+ indicators like GCaMP6f allow functional characterization by optical recordings. Here we report a turnkey microscopy system that enables simultaneous Ca2+ imaging at two parallel focal planes from intact DRG. By using consumer-grade optical components, the microscopy system is cost-effective and can be made broadly available without loss of capacity. It records low-intensity fluorescent signals at a wide field of view (1.9 × 1.3 mm) to cover a whole mouse DRG, with a high pixel resolution of 0.7 micron/pixel, a fast frame rate of 50 frames/sec, and the capability of remote focusing without perturbing the sample. The wide scanning range (100 mm) of the motorized sample stage allows convenient recordings of multiple DRGs in thoracic, lumbar, and sacral vertebrae. As a demonstration, we characterized mechanical neural encoding of visceral afferents innervating distal colon and rectum (colorectum) in GCaMP6f mice driven by VGLUT2 promotor. A post-processing routine is developed for conducting unsupervised detection of visceral afferent responses from GCaMP6f recordings, which also compensates the motion artifacts caused by mechanical stimulation of the colorectum. The reported system offers a cost-effective solution for high-throughput recordings of visceral afferent activities from a large volume of DRG tissues. We anticipate a wide application of this microscopy system to expedite our functional understanding of visceral innervations.

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.

Autofocusing technologies for whole slide imaging and automated microscopy.

Whole slide imaging (WSI) has moved digital pathology closer to diagnostic practice in recent years. Due to the inherent tissue topography variability, accurate autofocusing remains a critical challenge for WSI and automated microscopy systems. The traditional focus map surveying method is limited in its ability to acquire a high degree of focus points while still maintaining high throughput. Real-time approaches decouple image acquisition from focusing, thus allowing for rapid scanning while maintaining continuous accurate focus. This work reviews the traditional focus map approach and discusses the choice of focus measure for focal plane determination. It also discusses various real-time autofocusing approaches including reflective-based triangulation, confocal pinhole detection, low-coherence interferometry, tilted sensor approach, independent dual sensor scanning, beam splitter array, phase detection, dual-LED illumination and deep-learning approaches. The technical concepts, merits and limitations of these methods are explained and compared to those of a traditional WSI system. This review may provide new insights for the development of high-throughput automated microscopy imaging systems that can be made broadly available and utilizable without loss of capacity.

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.

Wide-field, high-resolution lensless on-chip microscopy via near-field blind ptychographic modulation.

We report a novel lensless on-chip microscopy platform based on near-field blind ptychographic modulation. In this platform, 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 those of 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, addressing the positioning accuracy issue plaguing regular ptychographic experiments. In our implementation, we use a low-cost, DIY scanning stage to perform blind diffuser modulation. Precise mechanical scanning that is critical in conventional ptychography experiments is no longer needed in our setup. 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. We validate the imaging performance via in vitro cell cultures, transparent and stained tissue sections, and a thick biological sample. We show that the recovered quantitative phase map can be used to perform effective cell segmentation of a dense yeast culture. We also demonstrate 3D digital refocusing of the thick biological sample based on the recovered wavefront. The reported platform provides a cost-effective and turnkey solution for large field-of-view, high-resolution, and quantitative on-chip microscopy. It is adaptable for a wide range of point-of-care-, global-health-, and telemedicine-related 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.

Rapid and robust whole slide imaging based on LED-array illumination and color-multiplexed single-shot autofocusing.

BACKGROUND: Digital pathology is experiencing an exponential period of growth catalyzed by advancements in imaging hardware and progresses in machine learning. The use of whole slide imaging (WSI) for digital pathology has recently been cleared for primary diagnosis in the US. The demand for using frozen section procedure for rapid identification of cancerous tissue during surgery is another driving force for the development of WSI. A conventional WSI system scans the tissue slide to different positions and acquires the digital images. In a typical implementation, a focus map is created prior to the scanning process, leading to significant overhead time and a necessity for high positional accuracy of the mechanical system. The resulting cost of WSI system is often prohibitive for frozen section procedure during surgery. METHODS: We report a novel WSI scheme based on a programmable LED array for sample illumination. In between two regular brightfield image acquisitions, we acquire one additional image by turning on a red and a green LED for color multiplexed illumination. We then identify the translational shift of the red- and green-channel images by maximizing the image mutual information or cross-correlation. The resulting translational shift is used for dynamic focus correction in the scanning process. Since we track the differential focus during adjacent acquisitions, there is no positional repeatability requirement in our scheme. RESULTS: We demonstrate a prototype WSI platform with a mean focusing error of ~0.3 microns. Different from previous implementations, this prototype platform requires no focus map surveying, no secondary camera or additional optics, and allows for continuous sample motion in the focus tracking process. CONCLUSIONS: A programmable LED array can be used for color-multiplexed single-shot autofocusing in WSI. The reported scheme may enable the development of cost-effective WSI platforms without positional repeatability requirement. It may also provide a turnkey solution for other high-content microscopy applications.

Optical recording reveals topological distribution of functionally classified colorectal afferent neurons in intact lumbosacral DRG.

Neuromodulation as a non-drug alternative for managing visceral pain in irritable bowel syndrome (IBS) may target sensitized afferents of distal colon and rectum (colorectum), especially their somata in the dorsal root ganglion (DRG). Developing selective DRG stimulation to manage visceral pain requires knowledge of the topological distribution of colorectal afferent somata which are sparsely distributed in the DRG. Here, we implemented GCaMP6f to conduct high-throughput optical recordings of colorectal afferent activities in lumbosacral DRG, that is, optical electrophysiology. Using a mouse ex vivo preparation with distal colorectum and L5-S1 DRG in continuity, we recorded 791 colorectal afferents' responses to graded colorectal distension (15, 30, 40, and 60 mmHg) and/or luminal shear flow (20-30 mL/min), then functionally classified them into four mechanosensitive classes, and determined the topological distribution of their somata in the DRG. Of the 791 colorectal afferents, 90.8% were in the L6 DRG, 8.3% in the S1 DRG, and only 0.9% in the L5 DRG. L6 afferents had all four classes: 29% mucosal, 18.4% muscular-mucosal, 34% low-threshold (LT) muscular, and 18.2% high-threshold (HT) muscular afferents. S1 afferents only had three classes: 19.7% mucosal, 34.8% LT muscular, and 45.5% HT muscular afferents. All seven L5 afferents were HT muscular. In L6 DRG, somata of HT muscular afferents were clustered in the caudal region whereas somata of the other classes did not cluster in specific regions. Outcomes of this study can directly inform the design and improvement of next-generation neuromodulation devices that target the DRG to alleviate visceral pain in IBS patients.

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.

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.

Preparation, characterization and application of a protein hydrogel with rapid self-healing and unique autofluoresent multi-functionalities.

A smart hydrogel with dual self-healing and autofluoresent functionalities is presented. The protein hydrogel is fabricated by denaturing bovine serum albumin in a basic environment. Upon gelation, autofluorescence is induced and the protein hydrogel can be excited by a wide range of spectrum, ranging from 320 to 520 nm. It was also found that the as-prepared autofluorescent protein hydrogel possessed rapid and repetitive self-healing capability. Without any external stimulus, more than 90% recovery of the mechanical strength can be obtained within 10 min after destruction. Moreover, the as-prepared hydrogel exhibits excellent biocompatibility and cell attachment property after its pH adjustment to neutral pH, while both autofluorescence and self-healing properties were still retained. This study suggests a promising means to prepare multi-functional protein hydrogel with dual physicochemical functionalities, which holds great potential in biomedical related applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 81-91, 2019.

Digital, Rapid, Accurate, and Label-Free Enumeration of Viable Microorganisms Enabled by Custom-Built On-Glass-Slide Culturing Device and Microscopic Scanning.

Accurately measuring the number of viable microorganisms plays an essential role in microbiological studies. Since the conventional agar method of enumerating visible colonies is time-consuming and not accurate, efforts have been made towards overcoming these limitations by counting the invisible micro-colonies. However, none of studies on micro-colony counting was able to save significant time or provide accurate results. Herein, we developed an on-glass-slide cell culture device that enables rapid formation of micro-colonies on a 0.38 mm-thick gel film without suffering from nutrient and oxygen deprivation during bacteria culturing. Employing a phase contrast imaging setup, we achieved rapid microscopic scanning of micro-colonies within a large sample area on the thin film without the need of fluorescent staining. Using Escherichia coli (E. coli) as a demonstration, our technique was able to shorten the culturing time to within 5 h and automatically enumerate the micro-colonies from the phase contrast images. Moreover, this method delivered more accurate counts than the conventional visible colony counting methods. Due to these advantages, this imaging-based micro-colony enumeration technique provides a new platform for the quantification of viable microorganisms.

Terapixel hyperspectral whole-slide imaging via slit-array detection and projection.

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.

Transform- and multi-domain deep learning for single-frame rapid autofocusing in whole slide imaging.

A whole slide imaging (WSI) system has recently been approved for primary diagnostic use in the US. The image quality and system throughput of WSI is largely determined by the autofocusing process. Traditional approaches acquire multiple images along the optical axis and maximize a figure of merit for autofocusing. Here we explore the use of deep convolution neural networks (CNNs) to predict the focal position of the acquired image without axial scanning. We investigate the autofocusing performance with three illumination settings: incoherent Kohler illumination, partially coherent illumination with two plane waves, and one-plane-wave illumination. We acquire ~130,000 images with different defocus distances as the training data set. Different defocus distances lead to different spatial features of the captured images. However, solely relying on the spatial information leads to a relatively bad performance of the autofocusing process. It is better to extract defocus features from transform domains of the acquired image. For incoherent illumination, the Fourier cutoff frequency is directly related to the defocus distance. Similarly, autocorrelation peaks are directly related to the defocus distance for two-plane-wave illumination. In our implementation, we use the spatial image, the Fourier spectrum, the autocorrelation of the spatial image, and combinations thereof as the inputs for the CNNs. We show that the information from the transform domains can improve the performance and robustness of the autofocusing process. The resulting focusing error is ~0.5 µm, which is within the 0.8-µm depth-of-field range. The reported approach requires little hardware modification for conventional WSI systems and the images can be captured on the fly without focus map surveying. It may find applications in WSI and time-lapse microscopy. The transform- and multi-domain approaches may also provide new insights for developing microscopy-related deep-learning networks. We have made our training and testing data set (~12 GB) open-source for the broad research community.

Dual light-emitting diode-based multichannel microscopy for whole-slide multiplane, multispectral and phase imaging.

We report the development of a multichannel microscopy for whole-slide multiplane, multispectral and phase imaging. We use trinocular heads to split the beam path into 6 independent channels and employ a camera array for parallel data acquisition, achieving a maximum data throughput of approximately 1 gigapixel per second. To perform single-frame rapid autofocusing, we place 2 near-infrared light-emitting diodes (LEDs) at the back focal plane of the condenser lens to illuminate the sample from 2 different incident angles. A hot mirror is used to direct the near-infrared light to an autofocusing camera. For multiplane whole-slide imaging (WSI), we acquire 6 different focal planes of a thick specimen simultaneously. For multispectral WSI, we relay the 6 independent image planes to the same focal position and simultaneously acquire information at 6 spectral bands. For whole-slide phase imaging, we acquire images at 3 focal positions simultaneously and use the transport-of-intensity equation to recover the phase information. We also provide an open-source design to further increase the number of channels from 6 to 15. The reported platform provides a simple solution for multiplexed fluorescence imaging and multimodal WSI. Acquiring an instant focal stack without z-scanning may also enable fast 3-dimensional dynamic tracking of various biological samples.