Real-time microscopic imaging of moving organs at single-cell resolution represents a major challenge in studying complex biology in living systems. Motion of the tissue from the cardiac and ...respiratory cycles severely limits intravital microscopy by compromising ultimate spatial and temporal imaging resolution. However, significant recent advances have enabled single-cell resolution imaging to be achieved in vivo. In this protocol, we describe experimental procedures for intravital microscopy based on a combination of thoracic surgery, tissue stabilizers and acquisition gating methods, which enable imaging at the single-cell level in the beating heart in the mouse. Setup of the model is typically completed in 1 h, which allows 2 h or more of continuous cardiac imaging. This protocol can be readily adapted for the imaging of other moving organs, and it will therefore broadly facilitate in vivo high-resolution microscopy studies.
Innate immune cells recruited to inflammatory sites have short life spans and originate from the marrow, which is distributed throughout the long and flat bones. While bone marrow production and ...release of leukocyte increases after stroke, it is currently unknown whether its activity rises homogeneously throughout the entire hematopoietic system. To address this question, we employed spectrally resolved in vivo cell labeling in the murine skull and tibia. We show that in murine models of stroke and aseptic meningitis, skull bone marrow-derived neutrophils are more likely to migrate to the adjacent brain tissue than cells that reside in the tibia. Confocal microscopy of the skull-dura interface revealed myeloid cell migration through microscopic vascular channels crossing the inner skull cortex. These observations point to a direct local interaction between the brain and the skull bone marrow through the meninges.
Macrophages populate the healthy myocardium and, depending on their phenotype, may contribute to tissue homeostasis or disease. Their origin and role in diastolic dysfunction, a hallmark of cardiac ...aging and heart failure with preserved ejection fraction, remain unclear. Here we show that cardiac macrophages expand in humans and mice with diastolic dysfunction, which in mice was induced by either hypertension or advanced age. A higher murine myocardial macrophage density results from monocyte recruitment and increased hematopoiesis in bone marrow and spleen. In humans, we observed a parallel constellation of hematopoietic activation: circulating myeloid cells are more frequent, and splenic
F-FDG PET/CT imaging signal correlates with echocardiographic indices of diastolic dysfunction. While diastolic dysfunction develops, cardiac macrophages produce IL-10, activate fibroblasts, and stimulate collagen deposition, leading to impaired myocardial relaxation and increased myocardial stiffness. Deletion of IL-10 in macrophages improves diastolic function. These data imply expansion and phenotypic changes of cardiac macrophages as therapeutic targets for cardiac fibrosis leading to diastolic dysfunction.
Fluorescent proteins have become essential reporter molecules for studying life at the cellular and sub-cellular level, re-defining the ways in which we investigate biology. However, because of ...intense light scattering, most organisms and tissues remain inaccessible to current fluorescence microscopy techniques at depths beyond several hundred micrometres. We describe a multispectral opto-acoustic tomography technique capable of high-resolution visualization of fluorescent proteins deep within highly light-scattering living organisms. The method uses multiwavelength illumination over multiple projections combined with selective-plane opto-acoustic detection for artifact-free data collection. Accurate image reconstruction is enabled by making use of wavelength-dependent light propagation models in tissue. By performing whole-body imaging of two biologically important and optically diffuse model organisms, Drosophila melanogaster pupae and adult zebrafish, we demonstrate the facility to resolve tissue-specific expression of eGFP and mCherrry fluorescent proteins for precise morphological and functional observations in vivo.
Fluorochromes have become essential reporter molecules in biological research. We show that the depth-resolved distribution of fluorochromes in small animals can be imaged with 25 fmol sensitivity ...and 150 microm spatial resolution by means of multispectral photoacoustic imaging. The major advantage of the multispectral approach is the sensitive differentiation of chromophores and fluorochromes of interest based on self-reference measurements, as evidenced in this study by resolving a commonly used fluorochrome (Alexa Fluor 750) in mouse. The suggested method is well suited for enhancing visualization of functional and molecular information in vivo and longitudinally.
The bioactive plant diterpene oridonin displays important pharmacological activities and is widely used in traditional Chinese medicine; however, its molecular mechanism of action is still ...incompletely described. In vitro and in vivo data have demonstrated anti-tumor activity of oridonin and its ability to interfere with several cell pathways; however, presently only the molecular chaperone HSP70 has been identified as a direct potential target of this compound. Here, using a combination of different proteomic approaches, innovative Cellular Thermal Shift Assay (CETSA) experiments, and classical biochemical methods, we demonstrate that oridonin interacts with Nucleolin, effectively modulating the activity of this multifunctional protein. The ability of oridonin to target Nucleolin and/or HSP70 could account for the bioactivity profile of this plant diterpene. Recently, Nucleolin has attracted attention as a druggable target, as its diverse functions are implicated in pathological processes such as cancer, inflammation, and viral infection. However, up to now, no small molecule as Nucleolin binders has been reported, thus our finding represents the first evidence of Nucleolin modulation by a small inhibitor.
Real-time imaging of moving organs and tissues at microscopic resolutions represents a major challenge in studying the complex biology of live animals. Here we present a technique based on a novel ...stabilizer setup combined with a gating acquisition algorithm for the imaging of a beating murine heart at the single-cell level. The method allows serial in vivo fluorescence imaging of the beating heart in live mice in both confocal and nonlinear modes over the course of several hours. We demonstrate the utility of this technique for in vivo optical sectioning and dual-channel time-lapse fluorescence imaging of cardiac ischaemia. The generic method could be adapted to other moving organs and thus broadly facilitate in vivo microscopic investigations.
Knowledge of cardiomyocyte biology is limited by the lack of methods to interrogate single-cell physiology in vivo. Here we show that contracting myocytes can indeed be imaged with optical microscopy ...at high temporal and spatial resolution in the beating murine heart, allowing visualization of individual sarcomeres and measurement of the single cardiomyocyte contractile cycle. Collectively, this has been enabled by efficient tissue stabilization, a prospective real-time cardiac gating approach, an image processing algorithm for motion-artifact-free imaging throughout the cardiac cycle, and a fluorescent membrane staining protocol. Quantification of cardiomyocyte contractile function in vivo opens many possibilities for investigating myocardial disease and therapeutic intervention at the cellular level.
Intravital fluorescence microscopy, through extended penetration depth and imaging resolution, provides the ability to image at cellular and subcellular resolution in live animals, presenting an ...opportunity for new insights into in vivo biology. Unfortunately, physiological induced motion components due to respiration and cardiac activity are major sources of image artifacts and impose severe limitations on the effective imaging resolution that can be ultimately achieved in vivo. Here we present a novel imaging methodology capable of automatically removing motion artifacts during intravital microscopy imaging of organs and orthotopic tumors. The method is universally applicable to different laser scanning modalities including confocal and multiphoton microscopy, and offers artifact free reconstructions independent of the physiological motion source and imaged organ. The methodology, which is based on raw data acquisition followed by image processing, is here demonstrated for both cardiac and respiratory motion compensation in mice heart, kidney, liver, pancreas and dorsal window chamber.
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