Rapid Volumetric Imaging Research Articles

Three-Dimensional Analysis of the Maxillary Sinuses in Ancient Crania Dated to the V-VI Centuries BCE from Opi (Italy): Volumetric Measurements in Ancient Skulls from the Necropolis of Opi, Abruzzi, Italy.

Cone beam computed tomography (CBCT) can provide precise information about complex anatomical structures as it is characterised by rapid volumetric image acquisition with high resolution. The aim of this study was to provide measurements for 20 ancient skulls of the Samnite people found in the necropolis of Opi, a small and isolated mountain village in Abruzzo, a region in central Italy. All the images (left and right) of the 20 ancient skulls from Opi were acquired. All the data are the property of the Department of Innovative Technologies in Medicine and Dentistry of G. D'Annunzio University, Chieti, and different parameters (area and volume) were measured and evaluated. The mean and standard deviation of the facial measurements were also calculated. All the data were subjected to statistical analysis. CBCT scan data of 20 fossil skulls did not show significant values regarding the MS area and the volume between the right and left sides. In the ancient skulls, no difference was found between the right and left sides.

Super-sectioning with multi-sheet reversible saturable optical fluorescence transitions (RESOLFT) microscopy

Light-sheet fluorescence microscopy is an invaluable tool for four-dimensional biological imaging of multicellular systems due to the rapid volumetric imaging and minimal illumination dosage. However, it is challenging to retrieve fine subcellular information, especially in living cells, due to the width of the sheet of light (>1 μm). Here, using reversibly switchable fluorescent proteins (RSFPs) and a periodic light pattern for photoswitching, we demonstrate a super-resolution imaging method for rapid volumetric imaging of subcellular structures called multi-sheet RESOLFT. Multiple emission-sheets with a width that is far below the diffraction limit are created in parallel increasing recording speed (1–2 Hz) to provide super-sectioning ability (<100 nm). Our technology is compatible with various RSFPs due to its minimal requirement in the number of switching cycles and can be used to study a plethora of cellular structures. We track cellular processes such as cell division, actin motion and the dynamics of virus-like particles in three dimensions.

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.

Maxillary sinus in gender determination: a morphometric analysis using cone beam computed tomography.

Gender determination of skeletal remains is an important forensic procedure in the identification process. Maxillary sinus remains intact even when the skull and other bones may be badly disfigured. Cone beam computed tomography (CBCT) can provide precise information about complex anatomical structures, as it is characterized by rapid volumetric image acquisition with high resolution. The present study aimed to assess the accuracy of maxillary sinus measurements using cone beam computed tomography in gender determination and to develop a formula using discriminate function analysis. Bilateral maxillary sinus images (left and right) were acquired for 200 patients (100 females and 100 males) and different parameters (width, length, height, area, perimeter, and volume) were measured and evaluated. Mean and standard deviation of both maxillary sinuses measurements were calculated and compared. The data was subjected to discriminative statistical analysis and analyzed using an unpaired t-test. The difference between all these variables was statistically significant between males and females. Based on discriminant analysis, the most pronounced variable in the differentiation of gender groups was maxillary sinus height. Gender assessment was established correctly with an accuracy of 75% for females and 64% for males with an overall accuracy of 69.5%. Adding other independent variables to the model did not result in an improvement in overall accuracy. Cone beam computed tomography measurement of maxillary sinus can be used as an aid in forensic anthropology for gender determination.

Deep focus light-field camera for handheld 3D intraoral scanning using crosstalk-free solid immersion microlens arrays.

3D in vivo imaging techniques facilitate disease tracking and treatment, but bulky configurations and motion artifacts limit practical clinical applications. Compact light-field cameras with microlens arrays offer a feasible option for rapid volumetric imaging, yet their utilization in clinical practice necessitates an increased depth-of-field for handheld operation. Here, we report deep focus light-field camera (DF-LFC) with crosstalk-free solid immersion microlens arrays (siMLAs), allowing large depth-of-field and high-resolution imaging for handheld 3D intraoral scanning. The siMLAs consist of thin PDMS-coated microlens arrays and a metal-insulator-metal absorber to extend the focal length with low optical crosstalk and specular reflection. The experimental results show that the immersion of MLAs in PDMS increases the focal length by a factor of 2.7 and the transmittance by 5.6%-27%. Unlike conventional MLAs, the siMLAs exhibit exceptionally high f-numbers up to f/6, resulting in a large depth-of-field for light-field imaging. The siMLAs were fully integrated into an intraoral scanner to reconstruct a 3D dental phantom with a distance measurement error of 82 ± 41 μm during handheld operation. The DF-LFC offers a new direction not only for digital dental impressions with high accuracy, simplified workflow, reduced waste, and digital compatibility but also for assorted clinical endoscopy and microscopy.

Aberration Modeling in Deep Learning for Volumetric Reconstruction of Light‐Field Microscopy

AbstractOptical aberration is a crucial issue in optical microscopes, fundamentally constraining the achievable imaging performance. As a commonly encountered one, spherical aberration is introduced by the refractive index mismatches between samples and their surrounding environments, leading to problems like low contrast, blurring, and distortion in imaging. Light‐field microscopy (LFM) has recently emerged as a powerful tool for rapid volumetric imaging. The presence of spherical aberration in LFM will cause substantial changes of the point spread function (PSF) and thus greatly affects the imaging performance. Here, the aberration‐modeling view‐channel‐depth (AM‐VCD) network is proposed for LFM reconstruction, effectively mitigating the influence of severe spherical aberration. By quantitatively estimating the spherical aberration in advance and incorporating it into the network training, the AM‐VCD achieves aberration‐corrected high‐speed visualization of 3D processes with uniform spatial resolution and real‐time reconstruction speed. Without necessitating hardware modifications, this method provides a convenient way for directly observing the 3D dynamics of samples in solution. The capability of AM‐VCD under a large refractive index mismatch is demonstrated through volumetric imaging of a large‐scale fishbone of a largemouth bass. Furthermore, the capability of AM‐VCD in nearly 100 Hz volumetric imaging of neutrophil migration and a beating heart in living zebrafish is investigated.

Volumetric light sheet imaging with adaptive optics correction.

Light sheet microscopy has developed quickly over the past decades and become a popular method for imaging live model organisms and other thick biological tissues. For rapid volumetric imaging, an electrically tunable lens can be used to rapidly change the imaging plane in the sample. For larger fields of view and higher NA objectives, the electrically tunable lens introduces aberrations in the system, particularly away from the nominal focus and off-axis. Here, we describe a system that employs an electrically tunable lens and adaptive optics to image over a volume of 499 × 499 × 192 μm3 with close to diffraction-limited resolution. Compared to the system without adaptive optics, the performance shows an increase in signal to background ratio by a factor of 3.5. While the system currently requires 7s/volume, it should be straightforward to increase the imaging speed to under 1s per volume.

Rapid Cellular-Resolution Skin Imaging with Optical Coherence Tomography Using All-Glass Multifocal Metasurfaces.

Cellular-resolution optical coherence tomography (OCT) is a powerful tool offering noninvasive histology-like imaging. However, like other optical microscopy tools, a high numerical aperture (N.A.) lens is required to generate a tight focus, generating a narrow depth of field, which necessitates dynamic focusing and limiting the imaging speed. To overcome this limitation, we developed a metasurface platform that generates multiple axial foci, which multiplies the volumetric OCT imaging speed by offering several focal planes. This platform offers accurate and flexible control over the number, positions, and intensities of axial foci generated. All-glass metasurface optical elements 8 mm in diameter are fabricated from fused-silica wafers and implemented into our scanning OCT system. With a constant lateral resolution of 1.1 μm over all depths, the multifocal OCT triples the volumetric acquisition speed for dermatological imaging, while still clearly revealing features of stratum corneum, epidermal cells, and dermal-epidermal junctions and offering morphological information as diagnostic criteria for basal cell carcinoma. The imaging speed can be further improved in a sparse sample, e.g., 7-fold with a seven-foci beam. In summary, this work demonstrates the concept of metasurface-based multifocal OCT for rapid virtual biopsy, further providing insights for developing rapid volumetric imaging systems with high resolution and compact volume.

Feasibility of delivered dose reconstruction for MR-guided SBRT of pancreatic tumors with fast, real-time 3D cine MRI

Background and purposeIn MR-guided SBRT of pancreatic cancer, intrafraction motion is typically monitored with (interleaved) 2D cine MRI. However, tumor surroundings are often not fully captured in these images, and motion might be distorted by through-plane movement. In this study, the feasibility of highly accelerated 3D cine MRI to reconstruct the delivered dose during MR-guided SBRT was assessed. Materials and methodsA 3D cine MRI sequence was developed for fast, time-resolved 4D imaging, featuring a low spatial resolution that allows for rapid volumetric imaging at 430 ms. The 3D cines were acquired during the entire beam-on time of 23 fractions of online adaptive MR-guided SBRT for pancreatic tumors on a 1.5 T MR-Linac. A 3D deformation vector field (DVF) was extracted for every cine dynamic using deformable image registration. Next, these DVFs were used to warp the partial dose delivered in the time interval between consecutive cine acquisitions. The warped dose plans were summed to obtain a total delivered dose. The delivered dose was also calculated under various motion correction strategies. Key DVH parameters of the GTV, duodenum, small bowel and stomach were extracted from the delivered dose and compared to the planned dose. The uncertainty of the calculated DVFs was determined with the inverse consistency error (ICE) in the high-dose regions. ResultsThe mean (SD) relative (ratio delivered/planned) D99% of the GTV was 0.94 (0.06), and the mean (SD) relative D0.5cc of the duodenum, small bowel, and stomach were respectively 0.98 (0.04), 1.00 (0.07), and 0.98 (0.06). In the fractions with the lowest delivered tumor coverage, it was found that significant lateral drifts had occurred. The DVFs used for dose warping had a low uncertainty with a mean (SD) ICE of 0.65 (0.07) mm. ConclusionWe employed a fast, real-time 3D cine MRI sequence for dose reconstruction in the upper abdomen, and demonstrated that accurate DVFs, acquired directly from these images, can be used for dose warping. The reconstructed delivered dose showed only a modest degradation of tumor coverage, mostly attainable to baseline drifts. This emphasizes the need for motion monitoring and development of intrafraction treatment adaptation solutions, such as baseline drift corrections.

Dual-resonant scanning multiphoton microscope with ultrasound lens and resonant mirror for rapid volumetric imaging

A dual-resonant scanning multiphoton (DRSM) microscope incorporating a tunable acoustic gradient index of refraction lens with a resonant mirror is developed for high-speed volumetric imaging. In the proposed microscope, the pulse train signal of a femtosecond laser is used to trigger an embedded field programmable gate array to sample the multiphoton excited fluorescence signal at the rate of one pixel per laser pulse. It is shown that a frame rate of around 8000 Hz can be obtained in the x–z plane for an image region with a size of 256 × 80 pixels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 343 × 343 × 120 μm3 with an image size of 256 × 256 × 80 voxels. Moreover, a volumetric imaging rate of over 30 Hz can be obtained for a large image volume of 256 × 256 × 80 voxels, which represents 343 × 343 × 120 μm3 in field-of-view. The rapid volumetric imaging rate eliminates the aliasing effect for observed temporal frequencies lower than 15 Hz. The practical feasibility of the DRSM microscope is demonstrated by observing the mushroom bodies of a drosophila brain and performing 3D dynamic observations of moving 10-μm fluorescent beads.

Fast volumetric imaging with line-scan confocal microscopy by electrically tunable lens at resonant frequency.

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and usually suffers from a reduced acquisition rate, which is critical to properly document rapid neurophysiological dynamics. In this study, we implemented an electrically tunable lens (ETL) in the line-scan confocal microscopy (LSCM), enabling the volumetric acquisition at the rate of 20 frames per second with a maximum volume of interest of 315 × 315 × 80 µm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 µm and 90.4 ± 2.1 µm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth axial penetration by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of both cleared mouse brain ex vivo samples and in vivo brains. The current study showed a successful application of resonant ETL for constructing a high-performance 3D axially scanning LSCM (asLSCM) system. Such advances in rapid volumetric imaging would significantly enhance our understanding of various dynamic biological processes.

Wavefront engineered light needle microscopy for axially resolved rapid volumetric imaging.

Increasing the acquisition speed of three-dimensional volumetric images is important-particularly in biological imaging-to unveil the structural dynamics and functionalities of specimens in detail. In conventional laser scanning fluorescence microscopy, volumetric images are constructed from optical sectioning images sequentially acquired by changing the observation plane, limiting the acquisition speed. Here, we present a novel method to realize volumetric imaging from two-dimensional raster scanning of a light needle spot without sectioning, even in the traditional framework of laser scanning microscopy. Information from multiple axial planes is simultaneously captured using wavefront engineering for fluorescence signals, allowing us to readily survey the entire depth range while maintaining spatial resolution. This technique is applied to real-time and video-rate three-dimensional tracking of micrometer-sized particles, as well as the prompt visualization of thick fixed biological specimens, offering substantially faster volumetric imaging.

Rac1 activation can generate untemplated, lamellar membrane ruffles

BackgroundMembrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions.ResultsHere, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles.ConclusionsCombining LLSM and optogenetics enables superior spatial and temporal control for studying such dynamic mechanisms. Demonstrated here, the techniques implemented provide insight into the complex nature of the molecular interplay involved in dynamic actin machinery, revealing that Rac1 activation can generate untemplated, lamellar protrusions.

Recurrent neural network-based volumetric fluorescence microscopy

Volumetric imaging of samples using fluorescence microscopy plays an important role in various fields including physical, medical and life sciences. Here we report a deep learning-based volumetric image inference framework that uses 2D images that are sparsely captured by a standard wide-field fluorescence microscope at arbitrary axial positions within the sample volume. Through a recurrent convolutional neural network, which we term as Recurrent-MZ, 2D fluorescence information from a few axial planes within the sample is explicitly incorporated to digitally reconstruct the sample volume over an extended depth-of-field. Using experiments on C. elegans and nanobead samples, Recurrent-MZ is demonstrated to significantly increase the depth-of-field of a 63×/1.4NA objective lens, also providing a 30-fold reduction in the number of axial scans required to image the same sample volume. We further illustrated the generalization of this recurrent network for 3D imaging by showing its resilience to varying imaging conditions, including e.g., different sequences of input images, covering various axial permutations and unknown axial positioning errors. We also demonstrated wide-field to confocal cross-modality image transformations using Recurrent-MZ framework and performed 3D image reconstruction of a sample using a few wide-field 2D fluorescence images as input, matching confocal microscopy images of the same sample volume. Recurrent-MZ demonstrates the first application of recurrent neural networks in microscopic image reconstruction and provides a flexible and rapid volumetric imaging framework, overcoming the limitations of current 3D scanning microscopy tools.

Rapid mesoscale volumetric imaging of neural activity with synaptic resolution.

Imaging neurons and neural circuits over large volumes at high speed and subcellular resolution is a difficult task. Incorporating a Bessel focus module into a two-photon fluorescence mesoscope, we achieved rapid volumetric imaging of neural activity over mesoscale with synaptic resolution. We applied it to calcium imaging of entire dendritic spans of neurons as well as neural ensembles within multiple cortical regions over two hemispheres of the awake mouse brain.

The use of optical coherence tomography and convolutional neural networks to distinguish normal and abnormal oral mucosa.

Incomplete surgical resection of head and neck squamous cell carcinoma (HNSCC) is the most common cause of local HNSCC recurrence. Currently, surgeons rely on preoperative imaging, direct visualization, palpation and frozen section to determine the extent of tissue resection. It has been demonstrated that optical coherence tomography (OCT), a minimally invasive, nonionizing near infrared mesoscopic imaging modality can resolve subsurface differences between normal and abnormal head and neck mucosa. Previous work has utilized two-dimensional OCT imaging which is limited to the evaluation of small regions of interest generated frame by frame. OCT technology is capable of performing rapid volumetric imaging, but the capacity and expertise to analyze this massive amount of image data is lacking. In this study, we evaluate the ability of a retrained convolutional neural network to classify three-dimensional OCT images of head and neck mucosa to differentiate normal and abnormal tissues with sensitivity and specificity of 100% and 70%, respectively. This method has the potential to serve as a real-time analytic tool in the assessment of surgical margins.

Solid immersion meniscus lens (SIMlens) for open-top light-sheet microscopy.

Open-top light-sheet (OTLS) microscopy has been developed for rapid volumetric imaging of large pathology specimens. A challenge with OTLS microscopy is the transmission of oblique illumination and detection beams through a horizontal sample plate without introducing aberrations. Previous solutions prevented vertical sample movement, constrained the refractive index of the sample, and/or hindered multi-resolution imaging. Here we describe a solid immersion meniscus lens, a wavefront-matching element that suppresses aberrations when illumination and detection beam transition between air and various high-index immersion media, thereby enabling multi-resolution OTLS microscopy of specimens cleared with diverse protocols.

Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

Highly-accelerated volumetric brain examination using optimized wave-CAIPI encoding.

Rapid volumetric imaging protocols could better utilize limited scanner resources. To develop and validate an optimized 6-minute high-resolution volumetric brain MRI examination using Wave-CAIPI encoding. Prospective. Ten healthy subjects and 20 patients with a variety of intracranial pathologies. At 3 T, MPRAGE, T2 -weighted SPACE, SPACE FLAIR, and SWI were acquired at 9-fold acceleration using Wave-CAIPI and for comparison at 2-4-fold acceleration using conventional GRAPPA. Extensive simulations were performed to optimize the Wave-CAIPI protocol and minimize both g-factor noise amplification and potential T1 /T2 blurring artifacts. Moreover, refinements in the autocalibrated reconstruction of Wave-CAIPI were developed to ensure high-quality reconstructions in the presence of gradient imperfections. In a randomized and blinded fashion, three neuroradiologists assessed the diagnostic quality of the optimized 6-minute Wave-CAIPI exam and compared it to the roughly 3× slower GRAPPA accelerated protocol using both an individual and head-to-head analysis. A noninferiority test was used to test whether the diagnostic quality of Wave-CAIPI was noninferior to the GRAPPA acquisition, with a 15% noninferiority margin. Among all sequences, Wave-CAIPI achieved negligible g-factor noise amplification (gavg ≤ 1.04) and burring artifacts from T1 /T2 relaxation. Improvements of our autocalibration approach for gradient imperfections enabled increased robustness to gradient mixing imperfections in tilted-field of view (FOV) prescriptions as well as variations in gradient and analog-to-digital converter (ADC) sampling rates. In the clinical evaluation, Wave-CAIPI achieved similar mean scores when compared with GRAPPA (MPRAGE: ØW = 4.03, ØG = 3.97; T2 w SPACE: ØW = 4.00, ØG = 4.00; SPACE FLAIR: ØW = 3.97, ØG = 3.97; SWI: ØW = 3.93, ØG = 3.83) and was statistically noninferior (N = 30, P < 0.05 for all sequences). The proposed volumetric brain exam retained comparable image quality when compared with the much longer conventional protocol. 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;50:961-974.

Imaging whole nervous systems: insights into behavior from worms to fish.

The development of systems combining rapid volumetric imaging with three-dimensional tracking has enabled the measurement of brain-wide dynamics in freely behaving animals such as worms, flies, and fish. These advances provide an exciting opportunity to understand the organization of neural circuits in the context of voluntary and natural behaviors. In this Comment, we highlight recent progress in this burgeoning area of research.