High resolution tumor targeting in living mice by means of multispectral optoacoustic tomography

BACKGROUND: Tumor targeting is of high clinical and biological relevance, and major efforts have been made to develop molecular imaging technologies for visualization of the disease markers in tissue. Of particular interest is apoptosis which has a profound role within tumor development and has significant effect on cancer malignancy. METHODS: Herein, we report on targeting of phosphatidylserine-exposing cells within live tumor allograft models using a synthetic near infrared zinc(II)-dipicolylamine probe. Visualization of the probe biodistribution is performed with whole body multispectral optoacoustic tomography (MSOT) system and subsequently compared to results attained by planar and tomographic fluorescence imaging systems. RESULTS: Compared to whole body optical visualization methods, MSOT attains remarkably better imaging capacity by delivering high-resolution scans of both disease morphology and molecular function in real time. Enhanced resolution of MSOT clearly showed that the probe mainly localizes in the vessels surrounding the tumor, suggesting that its tumor selectivity is gained by targeting the phosphatidylserine exposed on the surface of tumor vessels. CONCLUSIONS: The current study demonstrates the high potential of MSOT to broadly impact the fields of tumor diagnostics and preclinical drug development

Cryoimaging-Microscopy Implementation for 3D Optical Imaging

The structures and biochemistry properties of biological tissues are mostly affected by diseases. The visualization of organ structure and biochemistry helps in early detection and progression monitoring of diseases. Although, 2D imaging has traditionally been used to gain information from the tissue, it does not accurately represent many of the structures and functions. There currently exists a need for sensitive and specific methods to show detailed information about the structure of the tissue with high resolution and in 3D. The potential advantage of the high resolution 3D images is the ability to accurately probe structural and biochemical properties of the tissue. Not only the changes in structure, but also the changes in temporal physiological responses affected by oxidative stress (OS) at cellular levels. Thus, it would be valuable to detect the cellular metabolic states, which play a key role in understanding the pathogenesis of the disease, and to develop instruments to detect high resolution 3D images of the tissue. The objective of this research is to develop a second generation fluorescence optical imaging instrument to image the cellular redox state in 3D, in control and diseases conditions. I have improved upon one of optical instrument, called cryoimager software and hardware wise to enable higher resolution images. This higher resolution imaging resembles the microscopy capability in cryo temperatures for high resolution 3D imaging. In conclusion, high resolution optical instrumentation combined with signal and image processing tools provide quantitative physiological and structural information of diseased tissue

True-color 3D rendering of human anatomy using surface-guided color sampling from cadaver cryosection image data: A practical approach Jon Jatsu Azkue

Three-dimensional computer graphics are increasingly used for scientific visualization and for communicating anatomical knowledge and data. This study presents a practical method to produce true-color 3D surface renditions of anatomical structures. The procedure involves extracting the surface geometry of the structure of interest from a stack of cadaver cryosection images, using the extracted surface as a probe to retrieve color information from cryosection data, and mapping sampled colors back onto the surface model to produce a true-color rendition. Organs and body parts can be rendered separately or in combination to create custom anatomical scenes. By editing the surface probe, structures of interest can be rendered as if they had been previously dissected or prepared for anatomical demonstration. The procedure is highly flexible and nondestructive, offering new opportunities to present and communicate anatomical information and knowledge in a visually realistic manner. The technical procedure is described, including freely available open-source software tools involved in the production process, and examples of color surface renderings of anatomical structures are provided

Intensity Based Non-rigid Registration of 3D Whole Mouse Optical and MR Image Volumes

Novel magnetic resonance (MR) imaging techniques can be validated using accurate co-registration with histology. Whole-animal histological sections allow for simultaneous analysis of multiple tissues, and may also aid in registration by providing contextual information and structural support to tissues which if isolated from the body would be difficult to register. This thesis explores the feasibility of co-registration between whole mouse histology with 3D MR images using an intermediate optical image volume acquired during tissue sectioning. Of the two transformations required for this approach, 3D co-registration of MR and optical images is more challenging to perform due to changes in contrast, slice orientation, and resolution between these modalities. Here, an automated non-rigid registration technique utilizing mutual information is proposed to accurately register 3D whole mouse optical and MR images as a first step towards automated registration of histology. Validation of this technique was accomplished through calculation of post-registration target registration error

Advancing fluorescent contrast agent recovery methods for surgical guidance applications

Fluorescence-guided surgery (FGS) utilizes fluorescent contrast agents and specialized optical instruments to assist surgeons in intraoperatively identifying tissue-specific characteristics, such as perfusion, malignancy, and molecular function. In doing so, FGS represents a powerful surgical navigation tool for solving clinical challenges not easily addressed by other conventional imaging methods. With growing translational efforts, major hurdles within the FGS field include: insufficient tools for understanding contrast agent uptake behaviors, the inability to image tissue beyond a couple millimeters, and lastly, performance limitations of currently-approved contrast agents in accurately and rapidly labeling disease. The developments presented within this thesis aim to address such shortcomings. Current preclinical fluorescence imaging tools often sacrifice either 3D scale or spatial resolution. To address this gap in high-resolution, whole-body preclinical imaging tools available, the crux of this work lays on the development of a hyperspectral cryo-imaging system and image-processing techniques to accurately recapitulate high-resolution, 3D biodistributions in whole-animal experiments. Specifically, the goal is to correct each cryo-imaging dataset such that it becomes a useful reporter for whole-body biodistributions in relevant disease models. To investigate potential benefits of seeing deeper during FGS, we investigated short-wave infrared imaging (SWIR) for recovering fluorescence beyond the conventional top few millimeters. Through phantom, preclinical, and clinical SWIR imaging, we were able to 1) validate the capability of SWIR imaging with conventional NIR-I fluorophores, 2) demonstrate the translational benefits of SWIR-ICG angiography in a large animal model, and 3) detect micro-dose levels of an EGFR-targeted NIR-I probe during a Phase 0 clinical trial. Lastly, we evaluated contrast agent performances for FGS glioma resection and breast cancer margin assessment. To evaluate glioma-labeling performance of untargeted contrast agents, 3D agent biodistributions were compared voxel-by-voxel to gold-standard Gd-MRI and pathology slides. Finally, building on expertise in dual-probe ratiometric imaging at Dartmouth, a 10-pt clinical pilot study was carried out to assess the technique’s efficacy for rapid margin assessment. In summary, this thesis serves to advance FGS by introducing novel fluorescence imaging devices, techniques, and agents which overcome challenges in understanding whole-body agent biodistributions, recovering agent distributions at greater depths, and verifying agents’ performance for specific FGS applications

Quantitative Optical Imaging of Metabolic and Structural Biomarkers in Rodent Injury Models

The assessment of organ metabolic function using optical imaging techniques is an overgrowing field of disease diagnosis. The broad research objective of my PhD thesis is to detect quantitative biomarkers by developing and applying optical imaging and image processing tools to animal models of human diseases. To achieve this goal, I have designed and implemented an optical imaging instrument called in vivo fluorescence imager to study wound healing progress. I have also developed a 3-dimensional (3D) vascular segmentation technique that uses intrinsic fluorescence images of whole organs. Intrinsic fluorophores (autofluorescence signals) provide information about the status of cellular bioenergetics in different tissue types. Reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) are two key Krebs cycle coenzymes in mitochondria, which are autofluorescent. The ratio of these two fluorophores (NADH/FAD) is used as an optical biomarker for mitochondrial redox state of the tissues. The custom-designed optical tools have enabled me to probe the metabolic state of diseases as well as structural information of the organs at different regimes (in vivo, at cryogenic temperature, and in vitro). Here are the main projects that I have conducted and significantly contributed to: 1) Fluorescent metabolic imaging. I have designed and implemented an in vivo fluorescence imaging device to study diabetic wounds in small animals. This device can monitor the dynamics of the metabolism of the skin by capturing the images of the surface fluorescence of NADH and FAD. The area of the wounds can also be monitored simultaneously. The spatiotemporal mitochondrial redox ratio changes can give information on the status of wound healing online. This device was utilized to study diabetic wounds and the effect of photo-biomodulation on the wound healing progress. I have also utilized the optical cryo-imaging system to study the three-dimensional (3D) mitochondrial redox state of kidneys, hearts, livers, and wound biopsies of the small animal models of various injuries. For example, cryo-imaging was conducted on irradiated rat hearts during ischemia-reperfusion (IR) to investigate the role of mitochondrial metabolism in the differential susceptibility to IR injury. Also, I developed a 3D image processing tool that can segment and quantify the medullary versus the cortical redox state in the kidneys of animal injury models. 2) 3D Vascular-Metabolic Imaging (VMI). I have designed VMI, an image processing algorithm that segments vascular networks from intrinsic fluorescence. VMI allows the simultaneous acquisition of vasculature and metabolism in multiple organs. I demonstrate that this technique provides the vascular network of the whole organ without the need for a contrast agent. A proof validation has performed using TdTomato fluorescence expressing endothelium. The VMI also showed convincing evidence for the “minimum work” hypothesis in the vascular network by following Murray’s law. For a proof-of-concept, I have also utilized a partial body irradiation model that VMI can provide information on radiation-induced vascular regression. 3) Time-lapse fluorescence microscopy. I have utilized fluorescence microscopy to quantify the dynamics of cellular reactive oxygen species (ROS) concentration. ROS is imaged and quantified under oxygen or metabolic stress conditions in cells in vitro. This approach enabled me to study the sensitivity of retinal endothelial cells and pericytes to stress under high glucose conditions. In short, I developed and utilized optical bio-instrumentation and image processing tools to be able to detect metabolic and vascular information about different diseases

Optimization of biomarkers for morphological analysis of healthy and preeclamptic term human placental tissue sections using advanced fluorescence microscopy methods

Preeclampsia (PE) is a pregnancy-related disorder affecting 5-8% of women worldwide (4% in Norway). It is believed that placental ischemia is the initial event in the development of PE and is characterized by placental insufficiency and clinical symptoms such as hypertension and proteinuria. In this project, we aimed to study suitable biomarkers for morphological analysis of human term placenta from normal pregnancies and women with PE using advanced fluorescence microscopes. To reach this objective, we optimized the labeling steps for advanced fluorescence optical microscopy imaging of formalin-fixed paraffin-embedded (FFPE) and cryo-preserved tissue sections of the human placenta. Furthermore, morphological and subcellular differences between healthy and preeclamptic placentas were investigated. For this, various fluorescence microscopy techniques were explored, including whole-slide scanner, high-resolution deconvolution microscopy (DV) and super-resolution structured illumination microscopy (SIM), along with diverse image processing tools and analysis of the microscopy images. In this thesis, diverse strategies were examined for the labeling of placental biomarkers including immunofluorescence staining of laeverin, cytokeratin-7 (CK-7) and placental alkaline phosphatase (PLAP), as well as direct labeling of F-actin, membranes and nuclei via phalloidin-Atto 647 N, CellMask Orange (CMO) and DAPI, respectively. The microscopy investigation revealed actin spots abundantly localized in subtypes of the chorionic villi in both healthy and PE placentas, such as terminal villi (p-value 0.55), mature intermediate villi (p-value 0.50), immature intermediate villi (p-value 0.54) and stem villi (p-value 0.47), thus no observable differences. However, we found significant differences (p-value 0.015) of syncytial knots in PE compared to healthy tissue. A disorganized brushborder at the apical surface seems to be observed in the PE chorionic villi. Moreover, we found PLAP expression in the syncytial microvesicles in healthy placentas. The immunofluorescence study using laeverin and CK-7 antibodies seem to show co-localization in the syncytial plasma membrane in healthy placentas, though the labeled PE tissues showed laeverin expression in the syncytial plasma membrane and cytoplasm, including overexpression of laeverin in the fetal capillaries. In conclusion, the biomarkers explored in this study may have the potential to play an important role in understanding and predict PE in the future

Advances in cellular and sub-cellular level localization of lipids and metabolites using two- and three dimensional high-spatial resolution MALDI mass spectrometry imaging

This thesis presents efforts in the advancement and application of high-spatial resolution matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) for the mapping of small metabolites and lipids at the cellular and sub-cellular level. The following work presents a number of advances, using both 2- and 3-dimensional MALDI-MSI to enable visualization at the sub-cellular level. The first chapter consists of a general introduction to the technique of MALDI-MSI, and the seventh and final chapter provides a brief summary of the presented work and possible future directions. The second chapter presents a technology development for the optimization and application of matrix recrystallization to improve lipid ion signals in maize embryos and leaves. Using the optimized recrystallization conditions, the ion signals were improved three times, enhancing the image quality of lipid species with no apparent changes in their localization. Additionally, when methanol was used as a recrystallization solvent, unexpected side reactions were observed between phosphatidic acid and methanol vapor, suggesting recrystallization solvent should be carefully selected to avoid side reactions. The third chapter presents an application using 5- and 10-um high spatial resolution MALDI-MSI to explore quantitative fatty acyl distributions of two classes of thylakoid membrane lipids along the developmental gradient of maize leaves in two inbred lines, B73 and Mo17, and the reciprocal hybrid lines, B73xMo17 and Mo17xB73. This study demonstrated that high-resolution MALDI-MSI analysis can be directly applied to multicellular plant tissues to uncover cell-specific metabolic biology that has not been possible using traditional metabolomics methodologies. For example, certain thylakoid membrane lipids (e.g. phosphatidylglycerol (PG) 32:0) show genotype-specific differences in cellular distributions. Inbred B73 shows preferential localization of PG 32:0 in bundle sheath cells, while a more uniform distribution between bundle sheath and mesophyll cells in inbred Mo17. The fourth chapter present the first time MALDI-MSI has been applied for three dimensional chemical imaging of a single cell using newly fertilized individual zebrafish embryos as a model system. High-spatial resolution MALDI-MSI was used to map and visualize the three-dimensional spatial distribution of phospholipid classes, phosphatidylcholines (PC), phosphatidylethanolamines (PE), and phosphatidylinositols (PI), in the zebrafish embryo. The 3D MALDI-MSI volumetric reconstructions were then used to compare four different normalization approaches to find reliable relative quantification in 2D- and 3D- MALDI MSI data sets. Furthermore, two-dimensional MSI was studied for embryos at different cell developmental stages (1-, 2-, 4-, 8-, and 16-cell stage) to investigate the localization changes of some lipids, revealing heterogeneous localizations of different classes of lipids in the embryo. The fifth chapter discusses the development of a high-throughput MALDI-MS based metabolomics platform using a microarray of nanoparticles and organic matrices. Five matrices that provide broad metabolite coverage were selected and used to analyze turkey gut microbiome samples. Over two thousand unique metabolite features were reproducibly detected across intestinal samples from turkeys fed a diet amended with therapeutic or sub-therapeutic antibiotics, or non-amended feed. This protocol was applied to fifty two turkey cecal samples at three different time points from the antibiotic feed trial, which allowed distinct metabolite profiles to be discovered. The sixth chapter presents an on-tissue chemical modification strategy for high-spatial resolution MALDI-MSI. A mass spectrometry imaging methodology was use to selectively enhance the metabolite signals for a sub-metabolome at a time by performing on tissue derivatizations. Three well-known on-tissue derivatization methods were used: coniferyl aldehyde for primary amines, Girard’s reagent T for carbonyl groups, and 2-picolylamine for carboxylic acids. This proof of concept experiment was applied to cross-sections of maize leaves and roots, and enabled the identification of over five hundred new unique metabolite features. Combined, this approach facilitated the visualization of various classes of compounds, which can eventually allow high-spatial resolution MSI in the metabolomics scale

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

AbstractSoft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant

Soft X-Ray Tomography Reveals Gradual Chromatin Compaction and Reorganization during Neurogenesis In Vivo

SummaryThe realization that nuclear distribution of DNA, RNA, and proteins differs between cell types and developmental stages suggests that nuclear organization serves regulatory functions. Understanding the logic of nuclear architecture and how it contributes to differentiation and cell fate commitment remains challenging. Here, we use soft X-ray tomography (SXT) to image chromatin organization, distribution, and biophysical properties during neurogenesis in vivo. Our analyses reveal that chromatin with similar biophysical properties forms an elaborate connected network throughout the entire nucleus. Although this interconnectivity is present in every developmental stage, differentiation proceeds with concomitant increase in chromatin compaction and re-distribution of condensed chromatin toward the nuclear core. HP1β, but not nucleosome spacing or phasing, regulates chromatin rearrangements because it governs both the compaction of chromatin and its interactions with the nuclear envelope. Our experiments introduce SXT as a powerful imaging technology for nuclear architecture