Segmentation of Vascular Structures and Hematopoietic Cells in 3-D Microscopy Images and Quantitative Analysis

In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery

Visualization and Correction of Automated Segmentation, Tracking and Lineaging from 5-D Stem Cell Image Sequences

Results: We present an application that enables the quantitative analysis of multichannel 5-D (x, y, z, t, channel) and large montage confocal fluorescence microscopy images. The image sequences show stem cells together with blood vessels, enabling quantification of the dynamic behaviors of stem cells in relation to their vascular niche, with applications in developmental and cancer biology. Our application automatically segments, tracks, and lineages the image sequence data and then allows the user to view and edit the results of automated algorithms in a stereoscopic 3-D window while simultaneously viewing the stem cell lineage tree in a 2-D window. Using the GPU to store and render the image sequence data enables a hybrid computational approach. An inference-based approach utilizing user-provided edits to automatically correct related mistakes executes interactively on the system CPU while the GPU handles 3-D visualization tasks. Conclusions: By exploiting commodity computer gaming hardware, we have developed an application that can be run in the laboratory to facilitate rapid iteration through biological experiments. There is a pressing need for visualization and analysis tools for 5-D live cell image data. We combine accurate unsupervised processes with an intuitive visualization of the results. Our validation interface allows for each data set to be corrected to 100% accuracy, ensuring that downstream data analysis is accurate and verifiable. Our tool is the first to combine all of these aspects, leveraging the synergies obtained by utilizing validation information from stereo visualization to improve the low level image processing tasks.Comment: BioVis 2014 conferenc

Tracking Strain-Specific Morphogenesis and Angiogenesis of Murine Calvaria with Large-Scale Optoacoustic and Ultrasound Microscopy

Skull bone development is a dynamic and well-coordinated process playing a key role in maturation and maintenance of the bone marrow (BM), fracture healing, and progression of diseases such as osteoarthritis or osteoporosis. At present, dynamic transformation of the growing bone (osteogenesis) as well as its vascularization (angiogenesis) remain largely unexplored due to the lack of suitable in vivo imaging techniques capable of noninvasive visualization of the whole developing calvaria at capillary-level resolution. We present a longitudinal study on skull bone development using ultrasound-aided large-scale optoacoustic microscopy (U-LSOM). Skull bone morphogenesis and microvascular growth patterns were monitored in three common mouse strains (C57BL/6J, CD-1, and Athymic Nude-Foxn1nu) at the whole-calvaria scale over a 3-month period. Strain-specific differences in skull development were revealed by quantitative analysis of bone and vessel parameters, indicating the coupling between angiogenesis and osteogenesis during skull bone growth in a minimally invasive and label-free manner. The method further enabled identifying BM-specific sinusoidal vessels, and superficial skull vessels penetrating into BM compartments. Our approach furnishes a new high-throughput longitudinal in vivo imaging platform to study morphological and vascular skull alterations in health and disease, shedding light on the critical links between blood vessel formation, skull growth, and regeneration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress. Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific "niche" cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment

Molecular Imaging

The present book gives an exceptional overview of molecular imaging. Practical approach represents the red thread through the whole book, covering at the same time detailed background information that goes very deep into molecular as well as cellular level. Ideas how molecular imaging will develop in the near future present a special delicacy. This should be of special interest as the contributors are members of leading research groups from all over the world

B and T cell acute lymphoblastic leukemia evade chemotherapy at distinct sites in the bone marrow

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support treatment escape. Using 3D fluorescence imaging of 10 primary ALL xenografts we identify sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detect B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon short-term chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment

Modality Attention and Sampling Enables Deep Learning with Heterogeneous Marker Combinations in Fluorescence Microscopy

Fluorescence microscopy allows for a detailed inspection of cells, cellular networks, and anatomical landmarks by staining with a variety of carefully-selected markers visualized as color channels. Quantitative characterization of structures in acquired images often relies on automatic image analysis methods. Despite the success of deep learning methods in other vision applications, their potential for fluorescence image analysis remains underexploited. One reason lies in the considerable workload required to train accurate models, which are normally specific for a given combination of markers, and therefore applicable to a very restricted number of experimental settings. We herein propose Marker Sampling and Excite, a neural network approach with a modality sampling strategy and a novel attention module that together enable ($i$) flexible training with heterogeneous datasets with combinations of markers and ($ii$) successful utility of learned models on arbitrary subsets of markers prospectively. We show that our single neural network solution performs comparably to an upper bound scenario where an ensemble of many networks is na\"ively trained for each possible marker combination separately. In addition, we demonstrate the feasibility of our framework in high-throughput biological analysis by revising a recent quantitative characterization of bone marrow vasculature in 3D confocal microscopy datasets. Not only can our work substantially ameliorate the use of deep learning in fluorescence microscopy analysis, but it can also be utilized in other fields with incomplete data acquisitions and missing modalities.Comment: 17 pages, 5 figures, 3 pages supplement (3 figures

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

Optical imaging of the small intestine immune compartment across scales.

The limitations of 2D microscopy constrain our ability to observe and understand tissue-wide networks that are, by nature, 3-dimensional. Optical projection tomography (OPT) enables the acquisition of large volumes (ranging from micrometres to centimetres) in various tissues. We present a multi-modal workflow for the characterization of both structural and quantitative parameters of the mouse small intestine. As proof of principle, we evidence its applicability for imaging the mouse intestinal immune compartment and surrounding mucosal structures. We quantify the volumetric size and spatial distribution of Isolated Lymphoid Follicles (ILFs) and quantify the density of villi throughout centimetre-long segments of intestine. Furthermore, we exhibit the age and microbiota dependence for ILF development, and leverage a technique that we call reverse-OPT for identifying and homing in on regions of interest. Several quantification capabilities are displayed, including villous density in the autofluorescent channel and the size and spatial distribution of the signal of interest at millimetre-scale volumes. The concatenation of 3D imaging with reverse-OPT and high-resolution 2D imaging allows accurate localisation of ROIs and adds value to interpretations made in 3D. Importantly, OPT may be used to identify sparsely-distributed regions of interest in large volumes whilst retaining compatibility with high-resolution microscopy modalities, including confocal microscopy. We believe this pipeline to be approachable for a wide-range of specialties, and to provide a new method for characterisation of the mouse intestinal immune compartment