Volumetric chemical imaging by stimulated Raman projection microscopy and tomography.
ABSTRACT: Volumetric imaging allows global understanding of three-dimensional (3D) complex systems. Light-sheet fluorescence microscopy and optical projection tomography have been reported to image 3D volumes with high resolutions and at high speeds. Such methods, however, usually rely on fluorescent labels for chemical targeting, which could perturb the biological functionality in living systems. We demonstrate Bessel-beam-based stimulated Raman projection (SRP) microscopy and tomography for label-free volumetric chemical imaging. Our SRP microscope enables fast quantitation of chemicals in a 3D volume through a two-dimensional lateral scan. Furthermore, combining SRP and sample rotation, we demonstrate the SRP tomography that can reconstruct the 3D distribution of chemical compositions with optical spatial resolution at a higher speed than the Gaussian-beam-based stimulated Raman scattering sectioning imaging can. We explore the potential of our SRP technology by mapping polymer particles in 3D volumes and lipid droplets in adipose cells.
Project description:Even though in vivo imaging approaches have witnessed several new and important developments, specimens that exhibit high light scattering properties such as Drosophila melanogaster pupae are still not easily accessible with current optical imaging techniques, obtaining images only from subsurface features. This means that in order to obtain 3D volumetric information these specimens need to be studied either after fixation and a chemical clearing process, through an imaging window--thus perturbing physiological development -, or during early stages of development when the scattering contribution is negligible. In this paper we showcase how Optical Projection Tomography may be used to obtain volumetric images of the head eversion process in vivo in Drosophila melanogaster pupae, both in control and headless mutant specimens. Additionally, we demonstrate the use of Helical Optical Projection Tomography (hOPT) as a tool for high throughput 4D-imaging of several specimens simultaneously.
Project description:Three-dimensional visualization of tissue structures using optical microscopy facilitates the understanding of biological functions. However, optical microscopy is limited in tissue penetration due to severe light scattering. Recently, a series of tissue-clearing techniques have emerged to allow significant depth-extension for fluorescence imaging. Inspired by these advances, we develop a volumetric chemical imaging technique that couples Raman-tailored tissue-clearing with stimulated Raman scattering (SRS) microscopy. Compared with the standard SRS, the clearing-enhanced SRS achieves greater than 10-times depth increase. Based on the extracted spatial distribution of proteins and lipids, our method reveals intricate 3D organizations of tumor spheroids, mouse brain tissues, and tumor xenografts. We further develop volumetric phasor analysis of multispectral SRS images for chemically specific clustering and segmentation in 3D. Moreover, going beyond the conventional label-free paradigm, we demonstrate metabolic volumetric chemical imaging, which allows us to simultaneously map out metabolic activities of protein and lipid synthesis in glioblastoma. Together, these results support volumetric chemical imaging as a valuable tool for elucidating comprehensive 3D structures, compositions, and functions in diverse biological contexts, complementing the prevailing volumetric fluorescence microscopy.
Project description:Tomographic imaging using penetrating waves generates cross-sectional views of the internal anatomy of a living subject. For artefact-free volumetric imaging, projection views from a large number of angular positions are required. Here we show that a deep-learning model trained to map projection radiographs of a patient to the corresponding 3D anatomy can subsequently generate volumetric tomographic X-ray images of the patient from a single projection view. We demonstrate the feasibility of the approach with upper-abdomen, lung, and head-and-neck computed tomography scans from three patients. Volumetric reconstruction via deep learning could be useful in image-guided interventional procedures such as radiation therapy and needle biopsy, and might help simplify the hardware of tomographic imaging systems.
Project description:X-ray imaging techniques that capture variations in the x-ray phase can yield higher contrast images with lower x-ray dose than is possible with conventional absorption radiography. However, the extraction of phase information is often more difficult than the extraction of absorption information and requires a more sophisticated experimental arrangement. We here report a method for three-dimensional (3D) X-ray phase contrast computed tomography (CT) which gives quantitative volumetric information on the real part of the refractive index. The method is based on the recently developed X-ray speckle tracking technique in which the displacement of near field speckle is tracked using a digital image correlation algorithm. In addition to differential phase contrast projection images, the method allows the dark-field images to be simultaneously extracted. After reconstruction, compared to conventional absorption CT images, the 3D phase CT images show greatly enhanced contrast. This new imaging method has advantages compared to other X-ray imaging methods in simplicity of experimental arrangement, speed of measurement and relative insensitivity to beam movements. These features make the technique an attractive candidate for material imaging such as in-vivo imaging of biological systems containing soft tissue.
Project description:The compromise between lateral resolution and usable imaging depth range is a bottleneck for optical coherence tomography (OCT). Existing solutions for optical coherence microscopy (OCM) suffer from either large data size and long acquisition time or a nonideal point spread function. We present volumetric OCM of mouse brain <i>ex vivo</i> with a large depth coverage by leveraging computational adaptive optics (CAO) to significantly reduce the number of OCM volumes that need to be acquired with a Gaussian beam focused at different depths. We demonstrate volumetric reconstruction of <i>ex-vivo</i> mouse brain with lateral resolution of 2.2???m, axial resolution of 4.7???m, and depth range of ?1.2??mm optical path length, using only 11 OCT data volumes acquired on a spectral-domain OCM system. Compared to focus scanning with step size equal to the Rayleigh length of the beam, this is a factor of 4 fewer datasets required for volumetric imaging. Coregistered two-photon microscopy confirmed that CAO-OCM reconstructions can visualize various tissue microstructures in the brain. Our results also highlight the limitations of CAO in highly scattering media, particularly when attempting to reconstruct far from the focal plane or when imaging deep within the sample.
Project description:A precise volumetric assessment of maxillary alveolar defects in patients with cleft lip and palate can reduce donor site morbidity or allow accurate preparation of bone substitutes in future applications. However, there is a lack of agreement regarding the optimal volumetric technique to adopt. This study measured the alveolar bone defects by using two cone-beam computed tomography (CBCT)-based surgical simulation methods. Presurgical CBCT scans from 32 patients with unilateral or bilateral clefts undergoing alveolar bone graft surgery were analyzed. Two hands-on CBCT-based volumetric measurement methods were compared: the 3D real-scale printed model-based surgical method and the virtual surgical method. Different densities of CBCT were compared. Intra- and inter-examiner reliability was assessed. For patients with unilateral clefts, the average alveolar defect volumes were 1.09 ± 0.24 and 1.09 ± 0.25 mL (p > 0.05) for 3D printing- and virtual-based models, respectively; for patients with bilateral clefts, they were 2.05 ± 0.22 and 2.02 ± 0.27 mL (p > 0.05), respectively. Bland-Altman analysis revealed that the methods were equivalent for unilateral and bilateral alveolar cleft defect assessment. No significant differences or linear relationships were observed between adjacent different densities of CBCT for model production to obtain the measured volumes. Intra- and inter-examiner reliability was moderate to good (intraclass correlation coefficient (ICC) > 0.6) for all measurements. This study revealed that the volume of unilateral and bilateral alveolar cleft defects can be equally quantified by 3D-printed and virtual surgical simulation methods and provides alveolar defect-specific volumes which can serve as a reference for planning and execution of alveolar bone graft surgery.
Project description:Three-dimensional (3D) bioimaging, visualization and data analysis are in strong need of powerful 3D exploration techniques. We develop virtual finger (VF) to generate 3D curves, points and regions-of-interest in the 3D space of a volumetric image with a single finger operation, such as a computer mouse stroke, or click or zoom from the 2D-projection plane of an image as visualized with a computer. VF provides efficient methods for acquisition, visualization and analysis of 3D images for roundworm, fruitfly, dragonfly, mouse, rat and human. Specifically, VF enables instant 3D optical zoom-in imaging, 3D free-form optical microsurgery, and 3D visualization and annotation of terabytes of whole-brain image volumes. VF also leads to orders of magnitude better efficiency of automated 3D reconstruction of neurons and similar biostructures over our previous systems. We use VF to generate from images of 1,107 Drosophila GAL4 lines a projectome of a Drosophila brain.
Project description:Volumetric optical microscopy approaches that enable acquisition of three-dimensional (3D) information from a biological sample are attractive for numerous non-invasive imaging applications. The unprecedented structural details that these techniques provide have helped in our understanding of different aspects of architecture of cells, tissues, and organ systems as they occur in their natural states. Nonetheless, the instrumentation for most of these techniques is sophisticated, bulky, and costly, and is less affordable to most laboratory settings. Several miniature imagers based on webcams or low-cost sensors featuring easy assembly have been reported, for in situ imaging of biological structures at low costs. However, they have not been able to achieve the ability of 3D imaging throughout the entire volumes for spatiotemporal analyses of the structural changes in these specimens. Here we present a miniaturized optical tomography (mini-Opto) platform for low-cost, volumetric characterization of engineered living systems through hardware optimizations as well as applications of an optimized algebraic algorithm for image reconstruction.
Project description:Positron emission tomography (PET) of lung tumors suffers from breathing-motion induced blurring. Respiratory-correlated PET ameliorates motion blurring and enables visualization of lung tumor functional uptake throughout the breathing cycle but has achieved limited clinical use in radiotherapy planning. In this work, the authors propose a process for generating a gated PET maximum intensity projection (MIP), a breathing-phase projection of the 4D image set comprising gated PET images, as a technique to quantitatively and efficiently incorporate respiratory-correlated PET information into radiotherapy treatment planning.4D-CT and respiratory-gated PET using [(18)F]fluorodeoxyglucose (FDG) were acquired of three patients with a total of four small (4-18 cc), clearly defined lower-lobe lung tumors. Internal target volumes (ITVs) for the lung tumors were generated by threshold-based segmentation of PET-MIP images and ungated PET images (ITV(PET-MIP) and ITV(3D-PET), respectively), and by manual contouring of CT-MIP and end-exhale and end-inhale phases of 4D-CT (ITV(CT-MIP)) by a radiation oncologist. Because of the sensitivity of tumor segmentation to threshold value, several different thresholds were tested for ITV generation, including 40%, 30%, and 20% of maximum standardized uptake value (SUV(max)) for FDG as well as absolute SUV thresholds of 2.5 and 3.0. The normalized overlap and relative volumes of ITV(PET-MIP) and ITV(3D-PET) with respect to ITV(CT-MIP) were compared. The images were also visually compared. ITV(CT-MIP) was considered a gold standard for these tumors with CT-visible morphology.The mean and standard deviation normalized overlap and relative volumes between ITV(PET-MIP) and ITV(CT-MIP) were 0.68 ± 0.07 and 1.07 ± 0.42, respectively, averaged over all four tumors and all five threshold values. The mean and standard deviation normalized overlap and relative volumes of ITV(3D-PET) and ITV(CT-MIP) were 0.47 ± 0.12 and 0.69 ± 0.56, respectively.PET-MIP images better match CT-MIP images for this sample of four small CT-visible tumors as compared to ungated PET images, based on the metrics of volumetric overlap and relative volumes as well as visual interpretation. The PET-MIP is a way to incorporate 4D-PET imaging into the process of lung tumor contouring that is time-efficient for the radiation oncologist and involves minimal effort to implement in treatment planning software, because it requires only a single PET image beyond contouring on CT alone.
Project description:Focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening recently entered clinical testing for targeted drug delivery to the brain. Sources of variability exist in the current procedures, motivating the development of real-time monitoring and control techniques to improve treatment safety and efficacy. Here we used three-dimensional (3D) transcranial microbubble imaging to calibrate FUS exposure levels for volumetric BBB opening. Methods: Using a sparse hemispherical transmit/receive ultrasound phased array, pulsed ultrasound was focused transcranially into the thalamus of rabbits during microbubble infusion and multi-channel 3D beamforming was performed online with receiver signals captured at the subharmonic frequency. Pressures were increased pulse-by-pulse until subharmonic activity was detected on acoustic imaging (psub), and tissue volumes surrounding the calibration point were exposed at 50-100%psub via rapid electronic beam steering. Results: Spatially-coherent subharmonic microbubble activity was successfully reconstructed transcranially in vivo during calibration sonications. Multi-point exposures induced volumetric regions of elevated BBB permeability assessed via contrast-enhanced magnetic resonance imaging (MRI). At exposure levels ?75%psub, MRI and histological examination occasionally revealed tissue damage, whereas sonications at 50%psub were performed safely. Substantial intra-grid variability of FUS-induced bioeffects was observed via MRI, prompting future development of multi-point calibration schemes for improved treatment consistency. Receiver array sparsity and sensor configuration had substantial impacts on subharmonic detection sensitivity, and are factors that should be considered when designing next-generation clinical FUS brain therapy systems. Conclusion: Our findings suggest that 3D subharmonic imaging can be used to calibrate exposure levels for safe FUS-induced volumetric BBB opening, and should be explored further as a method for cavitation-mediated treatment guidance.