Rapid 3D Imaging Research Articles

Rapid 3D imaging at cellular resolution for digital cytopathology with a multi-camera array scanner (MCAS)

Optical microscopy has long been the standard method for diagnosis in cytopathology. Whole slide scanners can image and digitize large sample areas automatically, but are slow, expensive and therefore not widely available. Clinical diagnosis of cytology specimens is especially challenging since these samples are both spread over large areas and thick, which requires 3D capture. Here, we introduce a new parallelized microscope for scanning thick specimens across extremely wide fields-of-view (54 × 72 mm2) at 1.2 and 0.6 μm resolutions, accompanied by machine learning software to rapidly assess these 16 gigapixel scans. This Multi-Camera Array Scanner (MCAS) comprises 48 micro-cameras closely arranged to simultaneously image different areas. By capturing 624 megapixels per snapshot, the MCAS is significantly faster than most conventional whole-slide scanners. We used this system to digitize entire cytology samples (scanning three entire slides in 3D in just several minutes) and demonstrate two machine learning techniques to assist pathologists: first, an adenocarcinoma detection model in lung specimens (0.73 recall); second, a slide-level classification model of lung smears (0.969 AUC).

Unlocking the potential of large-scale 3D imaging with tissue clearing techniques.

The three-dimensional (3D) anatomical structure of living organisms is intrinsically linked to their functions, yet modern life sciences have not fully explored this aspect. Recently, the combination of efficient tissue clearing techniques and light-sheet fluorescence microscopy (LSFM) for rapid 3D imaging has improved access to 3D spatial information in biological systems. This technology has found applications in various fields, including neuroscience, cancer research, and clinical histopathology, leading to significant insights. It allows imaging of entire organs or even whole bodies of animals and humans at multiple scales. Moreover, it enables a form of spatial omics by capturing and analyzing cellome information, which represents the complete spatial organization of cells. While current 3D imaging of cleared tissues has limitations in obtaining sufficient molecular information, emerging technologies such as multi-round tissue staining and super-multicolor imaging are expected to address these constraints. 3D imaging using tissue clearing and light-sheet microscopy thus offers a valuable research tool in the current and future life sciences for acquiring and analyzing large-scale biological spatial information.

Spherical echo-planar time-resolved imaging (sEPTI) for rapid 3D quantitative and susceptibility imaging.

To develop a 3D spherical EPTI (sEPTI) acquisition and a comprehensive reconstruction pipeline for rapid high-quality whole-brain submillimeter and QSM quantification. For the sEPTI acquisition, spherical k-space coverage is utilized with variable echo-spacing and maximum kx ramp-sampling to improve efficiency and signal incoherency compared to existing EPTI approaches. For reconstruction, an iterative rank-shrinking B0 estimation and odd-even high-order phase correction algorithms were incorporated into the reconstruction to better mitigate artifacts from field imperfections. A physics-informed unrolled network was utilized to boost the SNR, where 1-mm and 0.75-mm isotropic whole-brain imaging were performed in 45 and 90 s at 3 T, respectively. These protocols were validated through simulations, phantom, and in vivo experiments. Ten healthy subjects were recruited to provide sufficient data for the unrolled network. The entire pipeline was validated on additional five healthy subjects where different EPTI sampling approaches were compared. Two additional pediatric patients with epilepsy were recruited to demonstrate the generalizability of the unrolled reconstruction. sEPTI achieved 1.4 faster imaging with improved image quality and quantitative map precision compared to existing EPTI approaches. The B0 update and the phase correction provide improved reconstruction performance with lower artifacts. The unrolled network boosted the SNR, achieving high-quality and QSM quantification with single average data. High-quality reconstruction was also obtained in the pediatric patients using this network. sEPTI achieved whole-brain distortion-free multi-echo imaging and and QSM quantification at 0.75 mm in 90 s which has the potential to be useful for wide clinical applications.

Axially swept open-top light-sheet microscopy for densely labeled clinical specimens.

Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.

Phase-modulated multi-foci microscopy for rapid 3D imaging

3D imaging technology is pivotal in monitoring the functional dynamics and morphological alterations in living cells and tissues. However, conventional volumetric imaging associated with mechanical z-scanning encounters challenges in limited 3D imaging speed with inertial artifact. Here, we present a unique phase-modulated multi-foci microscopy (PM3) technique to achieve snapshot 3D imaging with the advantages of extended imaging depths and adjustable imaging intervals between each focus in a rapid fashion. To accomplish the tasks, we utilize a spatial light modulator (SLM) to encode the phases of the scattered or fluorescence light emanating from a volumetric sample and then project the multiple-depth images of the sample onto a single charge-coupled device camera for rapid 3D imaging. We demonstrate that the PM3 technique provides ∼55-fold improvement in imaging depth in polystyrene beads phantom compared to the depth of field of the objective lens used. PM3 also enables the real-time monitoring of Brownian motion of fluorescent beads in water at a 15 Hz volume rate. By precisely manipulating the phase of scattered light on the SLM, PM3 can pinpoint the specific depth information in living zebrafish and rapidly observe the 3D dynamic processes of blood flow in the zebrafish trunk. This work shows that the PM3 technique developed is robust and versatile for fast 3D dynamic imaging in biological and biomedical systems.

Graded arc beam in light needle microscopy for axially resolved, rapid volumetric imaging without nonlinear processes.

High-speed three-dimensional (3D) imaging is essential for revealing the structure and functions of biological specimens. Confocal laser scanning microscopy has been widely employed for this purpose. However, it requires a time-consuming image-stacking procedure. As a solution, we previously developed light needle microscopy using a Bessel beam with a wavefront-engineered approach [Biomed. Opt. Express13, 1702 (2022)10.1364/BOE.449329]. However, this method applies only to multiphoton excitation microscopy because of the requirement to reduce the sidelobes of the Bessel beam. Here, we introduce a beam that produces a needle spot while eluding the intractable artifacts due to the sidelobes. This beam can be adopted even in one-photon excitation fluorescence 3D imaging. The proposed method can achieve real-time, rapid 3D observation of 200-nm particles in water at a rate of over 50 volumes per second. In addition, fine structures, such as the spines of neurons in fixed mouse brain tissue, can be visualized in 3D from a single raster scan of the needle spot. The proposed method can be applied to various modalities in biological imaging, enabling rapid 3D image acquisition.

The Usefulness of a 3D Roadmap of Occluded Vessels Created from Rapid 3D Proton Density-Weighted Imaging for Mechanical Thrombectomy

The Usefulness of a 3D Roadmap of Occluded Vessels Created from Rapid 3D Proton Density-Weighted Imaging for Mechanical Thrombectomy

Rapid 3D isotropic imaging of whole organ with double-ring light-sheet microscopy and self-learning side-lobe elimination.

Bessel-like plane illumination forms a new type of light-sheet microscopy with ultra-long optical sectioning distance that enables rapid 3D imaging of fine cellular structures across an entire large tissue. However, the side-lobe excitation of conventional Bessel light sheets severely impairs the quality of the reconstructed 3D image. Here, we propose a self-supervised deep learning (DL) approach that can completely eliminate the residual side lobes for a double-ring-modulated non-diffraction light-sheet microscope, thereby substantially improving the axial resolution of the 3D image. This lightweight DL model utilizes the own point spread function (PSF) of the microscope as prior information without the need for external high-resolution microscopy data. After a quick training process based on a small number of datasets, the grown-up model can restore sidelobe-free 3D images with near isotropic resolution for diverse samples. Using an advanced double-ring light-sheet microscope in conjunction with this efficient restoration approach, we demonstrate 5-minute rapid imaging of an entire mouse brain with a size of ∼12 mm × 8 mm × 6 mm and achieve uniform isotropic resolution of ∼4 µm (1.6-µm voxel) capable of discerning the single neurons and vessels across the whole brain.

Advancing full-field metrology: rapid 3D imaging with geometric phase ferroelectric liquid crystal technology in full-field optical coherence microscopy.

Optical coherence microscopy (OCM) is a variant of OCT in which a high-numerical aperture lens is used. Full-field OCM (FF-OCM) is an emerging non-invasive, label-free, interferometric technique for imaging of surface structures or semi-transparent biomedical subjects with micron-scale resolutions. Different approaches to three dimensional full-field optical metrology are reviewed. The usual method for the phase-shifting technique in FF-OCM involves mechanically moving a mirror to change the optical path difference for obtaining en-face OCM images. However, with the use of a broadband source in FF-OCM, the phase shifts of different spectral components are not the same, resulting in the ambiguities in 3D image reconstruction. In this study, we demonstrate, by imaging tissues and cells, a unique geometric phase-shifter based on ferroelectric liquid crystal technology, to realize achromatic phase-shifting for rapid three-dimensional imaging in a FF-OCM system.

3D-EPI blip-up/down acquisition (BUDA) with CAIPI and joint Hankel structured low-rank reconstruction for rapid distortion-free high-resolution mapping.

This work aims to develop a novel distortion-free 3D-EPI acquisition and image reconstruction technique for fast and robust, high-resolution, whole-brain imaging as well as quantitative mapping. 3D Blip-up and -down acquisition (3D-BUDA) sequence is designed for both single- and multi-echo 3D gradient recalled echo (GRE)-EPI imaging using multiple shots with blip-up and -down readouts to encode B0 field map information. Complementary k-space coverage is achieved using controlled aliasing in parallel imaging (CAIPI) sampling across the shots. For image reconstruction, an iterative hard-thresholding algorithm is employed to minimize the cost function that combines field map information informed parallel imaging with the structured low-rank constraint for multi-shot 3D-BUDA data. Extending 3D-BUDA to multi-echo imaging permits mapping. For this, we propose constructing a joint Hankel matrix along both echo and shot dimensions to improve the reconstruction. Experimental results on in vivo multi-echo data demonstrate that, by performing joint reconstruction along with both echo and shot dimensions, reconstruction accuracy is improved compared to standard 3D-BUDA reconstruction. CAIPI sampling is further shown to enhance image quality. For mapping, parameter values from 3D-Joint-CAIPI-BUDA and reference multi-echo GRE are within limits of agreement as quantified by Bland-Altman analysis. The proposed technique enables rapid 3D distortion-free high-resolution imaging and mapping. Specifically, 3D-BUDA enables 1-mm isotropic whole-brain imaging in 22 s at 3T and 9s on a 7T scanner. The combination of multi-echo 3D-BUDA with CAIPI acquisition and joint reconstruction enables distortion-free whole-brain mapping in 47 s at 1.1 × 1.1 × 1.0 mm3 resolution.

Linearly modulated multi-focal diffractive lens for multi-sheet excitation of flow driven samples in a light-sheet fluorescence microscope

Light sheet fluorescence microscope with single light sheet illumination enables rapid 3D imaging of living cells. In this paper we show the design, fabrication and characterization of a diffractive optical element producing several light sheets along a 45° inclined tube. The element, which is based on a multi-focal diffractive lens and a linear grating, generates five thin light sheets with equal intensities when combined with a refractive cylindrical lens. The generated uniform light sheets can be applied for the scanning of samples in tubes enabling flow-driven 3-dimensional imaging.

Combined method of whole mount and block-face imaging: Acquisition of 3D data of gene expression pattern from conventional in situ hybridization.

Visualization of spatiotemporal expression of a gene of interest is a fundamental technique for analyzing the involvements of genes in organ development. In situ hybridization (ISH) is one of the most popular methods for visualizing gene expression. When conventional ISH is performed on sections or whole-mount specimens, the gene expression pattern is represented in 2-dimensional (2D) microscopic images or in the surface view of the specimen. To obtain 3-dimensional (3D) data of gene expression from conventional ISH, the "serial section method" has traditionally been employed. However, this method requires an extensive amount of time and labor because it requires researchers to collect a tremendous number of sections, label all sections by ISH, and image them before 3D reconstruction. Here, we proposed a rapid and low-cost 3D imaging method that can create 3D gene expression patterns from conventional ISH-labeled specimens. Our method consists of a combination of whole-mount ISH and Correlative Microscopy and Blockface imaging (CoMBI). The whole-mount ISH-labeled specimens were sliced using a microtome or cryostat, and all block-faces were imaged and used to reconstruct 3D images by CoMBI. The 3D data acquired using our method showed sufficient quality to analyze the morphology and gene expression patterns in the developing mouse heart. In addition, 2D microscopic images of the sections can be obtained when needed. Correlating 2D microscopic images and 3D data can help annotate gene expression patterns and understand the anatomy of developing organs. These results indicated that our method can be useful in the field of developmental biology.

High contrast, isotropic, and uniform 3D-imaging of centimeter-scale scattering samples using structured illumination light-sheet microscopy with axial sweeping.

Light-sheet fluorescent microscopy (LSFM) has, in recent years, allowed for rapid 3D-imaging of cleared biomedical samples at larger and larger scale. However, even in cleared samples, multiple light scattering often degrades the imaging contrast and widens the optical sectioning. Accumulation of scattering intensifies these negative effects as light propagates inside the tissue, which accentuates the issues when imaging large samples. With axially swept light-sheet microscopy (ASLM), centimeter-scale samples can be scanned with a uniform micrometric optical sectioning. But to fully utilize these benefits for 3D-imaging in biomedical tissue samples, suppression of scattered light is needed. Here, we address this by merging ASLM with light-sheet based structured illumination into Structured Illumination Light-sheet Microscopy with Axial Sweeping (SILMAS). The SILMAS method thus enables high-contrast imaging, isotropic micrometric resolution and uniform optical sectioning in centimeter-scale scattering samples, creating isotropic 3D-volumes of e.g., whole mouse brains without the need for any computation-heavy post-processing. We demonstrate the effectiveness of the approach in agarose gel phantoms with fluorescent beads, and in an PFF injected alpha-synuclein transgenic mouse model tagged with a green fluorescent protein (SynGFP). SILMAS imaging is compared to standard ASLM imaging on the same samples and using the same optical setup, and is shown to increase contrast by as much as 370% and reduce widening of optical sectioning by 74%. With these results, we show that SILMAS improves upon the performance of current state-of-the-art light-sheet microscopes for large and imperfectly cleared tissue samples and is a valuable addition to the LSFM family.

Rapid high-resolution volumetric imaging via laser ablation delayering and confocal imaging

Acquiring detailed 3D images of samples is needed for conducting thorough investigations in a wide range of applications. Doing so using nondestructive methods such as X-ray computed tomography (X-ray CT) has resolution limitations. Destructive methods, which work based on consecutive delayering and imaging of the sample, face a tradeoff between throughput and resolution. Using focused ion beam (FIB) for delayering, although high precision, is low throughput. On the other hand, mechanical methods that can offer fast delayering, are low precision and may put the sample integrity at risk. Herein, we propose to use femtosecond laser ablation as a delayering method in combination with optical and confocal microscopy as the imaging technique for performing rapid 3D imaging. The use of confocal microscopy provides several advantages. First, it eliminates the 3D image distortion resulting from non-flat layers, caused by the difference in laser ablation rate of different materials. It further allows layer height variations to be maintained within a small range. Finally, it enables material characterization based on the processing of material ablation rate at different locations. The proposed method is applied on a printed circuit board (PCB), and the results are validated and compared with the X-ray CT image of the PCB part.

Rapid 3D nondestructive imaging technology for batteries: Photoacoustic microscopy

Rapid 3D nondestructive imaging technology for batteries: Photoacoustic microscopy

Error analysis and 3D reconstruction using airborne array InSAR images

Multi-temporal synthetic aperture radar interferometry (MT-InSAR) is able to detect surface deformation and reconstruct a 3D surface model with high precision but requires a long observation period to accumulate the multi-baseline SAR images. The airborne array InSAR system is able to acquire a stack of multi-baseline SAR images in a single acquisition, which significantly improves the 3D modeling capability. However, processing the images obtained by the low-altitude platform using the conventional model will lead to geometric approximation (GA) errors, such as flattened phase error and reference error, which degrade the precision of the 3D reconstruction. In this paper, we quantitatively analyze the error sources of the array-InSAR interferograms and design a hybrid 3D phase unwrapping approach for 3D reconstruction. A hypothesis test is developed to identify the phase ambiguity by comparing the initial solution of the least-square with that of the beam-forming. Three indicators are proposed to identify the reliable arcs, achieving a reliable phase unwrapping. The L1 norm approach is adopted to detect the unwrapping errors during the spatial unwrapping and a two-tie network strategy is used to process the data in the individual blocks from the perspective of global optimization. Furthermore, an iterative scheme is recommended to compensate the geometric approximation errors. The main advantage of the proposed algorithm is the compensation of the GA error and reliable phase unwrapping. The experimental results by both simulated and real SAR data show that the proposed algorithm can eliminate the GA error and provide a viable solution to rapid 3D SAR imaging with an airborne platform.

The use of 3-dimensional imaging of Holstein cows to estimate body weight and monitor the composition of body weight change throughout lactation

Three-dimensional (3D) imaging offers new possibilities in animal phenotyping. Here, we investigated how this technology can be used to study the morphological changes that occur in dairy cows over the course of a single lactation. First, we estimated the individual body weight (BW) of dairy cows using traits measured with 3D images. To improve the quality of prediction, we monitored body growth (via 3D imaging), gut fill (via individual dry matter intake), and body reserves (via body condition score) throughout lactation. A group of 16 Holstein cows-8 in their first lactation, 4 in their second lactation, and 4 in their third or higher lactation-was scanned in 3D once a month for an entire lactation. Values of morphological traits (e.g., chest depth or hip width) increased continuously with parity, but cows in their first lactation experienced the largest increase during the monitoring period. Values of partial volume, estimated from point of shoulder to pin bone, predicted BW with an error of 25.4 kg (R2 = 0.92), which was reduced to 14.3 kg when the individual effect of cows was added to the estimation model. The model was further improved by the addition of partial surface area (from point of shoulder to pin bone), hip width, chest depth, diagonal length, and heart girth, which increased the R2 of BW prediction to 0.94 and decreased root mean square error to 22.1 kg. The different slopes for individual cows were partly explained by body condition score and morphological traits, indicating that they may have reflected differences in body density among animals. Changes in BW over the course of lactation were mostly due to changes in growth, which accounted for around two-thirds of BW gain regardless of parity. Body reserves and gut fill had smaller but still notable effects on body composition, with a higher gain in body reserves and gut fill for cows in their first lactation compared with multiparous cows. This work demonstrated the potential for rapid and low-cost 3D imaging to facilitate the monitoring of several traits of high interest in dairy livestock farming.

Extended field-of-view light-sheet microscopy

Light-sheet fluorescence microscopy enables rapid 3D imaging of biological samples. Unlike confocal and two-photon microscopes, a light-sheet microscope illuminates the focal plane with an objective orthogonal to the detection axis and images it in a single snapshot. Its combination of high contrast and minimal sample exposure make it ideal to image thick samples with sub-cellular resolution. To uniformly illuminate a wide field-of-view without compromising axial resolution, propagation-invariant light-fields such as Bessel and Airy beams have been put forward. These beams do however irradiate the sample with a relatively broad transversal structure. The fluorescence excited by the side lobes of Bessel beams can be blocked physically during recording; though at the cost of increased sample exposure. In contrast, the Airy beam has a fine transversal structure that is both curved and asymmetric. Its fine structure captures all the high-frequency components that enable high axial resolution without the need to discard useful fluorescence. This advantage does not carry over naturally to two-photon excitation where the fine transversal structure is suppressed. We demonstrate a symmetric and planar Airy light-sheet that can be used with two-photon excitation and that does not rely on deconvolution.

Linearly modulated multi-focal diffractive lens for multi-sheet excitation of flow-driven samples in a light-sheet fluorescence microscope

Light-sheet fluorescence microscopy (LSFM) with single light-sheet illumination enables rapid 3Dimaging of living cells. In this paper we show the design, fabrication and characterization of a diffractive optical element producing several light sheets along an inclined tube for applications in flow-driven imaging. The element, which is based on a multi-focal Fresnel zone plate and a linear grating, generates in combination with a refractive cylindrical lens five thin light sheets of equal intensity.

Rapid 3D nanoscale coherent imaging via physics-aware deep learning

Phase retrieval, the problem of recovering lost phase information from measured intensity alone, is an inverse problem that is widely faced in various imaging modalities ranging from astronomy to nanoscale imaging. The current process of phase recovery is iterative in nature. As a result, the image formation is time consuming and computationally expensive, precluding real-time imaging. Here, we use 3D nanoscale X-ray imaging as a representative example to develop a deep learning model to address this phase retrieval problem. We introduce 3D-CDI-NN, a deep convolutional neural network and differential programing framework trained to predict 3D structure and strain, solely from input 3D X-ray coherent scattering data. Our networks are designed to be “physics-aware” in multiple aspects; in that the physics of the X-ray scattering process is explicitly enforced in the training of the network, and the training data are drawn from atomistic simulations that are representative of the physics of the material. We further refine the neural network prediction through a physics-based optimization procedure to enable maximum accuracy at lowest computational cost. 3D-CDI-NN can invert a 3D coherent diffraction pattern to real-space structure and strain hundreds of times faster than traditional iterative phase retrieval methods. Our integrated machine learning and differential programing solution to the phase retrieval problem is broadly applicable across inverse problems in other application areas.