RESUMEN
Anemia and iron deficiency continue to be the most prevalent nutritional disorders in the world, affecting billions of people in both developed and developing countries. The initial diagnosis of anemia is typically based on several markers, including red blood cell (RBC) count, hematocrit and total hemoglobin. Using modern hematology analyzers, erythrocyte parameters such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), etc. are also being used. However, most of these commercially available analyzers pose several disadvantages: they are expensive instruments that require significant bench space and are heavy enough to limit their use to a specific lab and lead to a delay in results, making them less practical as a point-of-care instrument that can be used for swift clinical evaluation. Thus, there is a need for a portable and economical hematology analyzer that can be used at the point of need. In this work, we evaluated the performance of a system referred to as the cell tracking velocimetry (CTV) to measure several hematological parameters from fresh human blood obtained from healthy donors and from sickle cell disease subjects. Our system, based on the paramagnetic behavior that deoxyhemoglobin or methemoglobin containing RBCs experience when suspended in water after applying a magnetic field, uses a combination of magnets and microfluidics and has the ability to track the movement of thousands of red cells in a short period of time. This allows us to measure not only traditional RBC indices but also novel parameters that are only available for analyzers that assess erythrocytes on a cell by cell basis. As such, we report, for the first time, the use of our CTV as a hematology analyzer that is able to measure MCV, MCH, mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), the percentage of hypochromic cells (which is an indicator of insufficient marrow iron supply that reflects recent iron reduction), and the correlation coefficients between these metrics. Our initial results indicate that most of the parameters measured with CTV are within the normal range for healthy adults. Only the parameters related to the red cell volume (primarily MCV and RDW) were outside the normal range. We observed significant discrepancies between the MCV measured by our technology (and also by an automated cell counter) and the manual method that calculates MCV through the hematocrit obtained by packed cell volume, which are attributed to the artifacts of plasma trapping and cell shrinkage. While there may be limitations for measuring MCV, this device offers a novel point of care instrument to provide rapid RBC parameters such as iron stores that are otherwise not rapidly available to the clinician. Thus, our CTV is a promising technology with the potential to be employed as an accurate, economical, portable and fast hematology analyzer after applying instrument-specific reference ranges or correction factors.
Asunto(s)
Anemia de Células Falciformes/sangre , Rastreo Celular/instrumentación , Índices de Eritrocitos , Citometría de Flujo/instrumentación , Microfluídica/instrumentación , Adulto , Estudios de Casos y Controles , Exactitud de los Datos , Recuento de Eritrocitos , Eritrocitos , Femenino , Hematócrito , Hemoglobinas/análisis , Humanos , Campos Magnéticos , Masculino , Persona de Mediana Edad , Valores de Referencia , Adulto JovenRESUMEN
Arabidopsis (Arabidopsis thaliana) primary and lateral roots (LRs) are well suited for 3D and 4D microscopy, and their development provides an ideal system for studying morphogenesis and cell proliferation dynamics. With fast-advancing microscopy techniques used for live-imaging, whole tissue data are increasingly available, yet present the great challenge of analyzing complex interactions within cell populations. We developed a plugin "Live Plant Cell Tracking" (LiPlaCeT) coupled to the publicly available ImageJ image analysis program and generated a pipeline that allows, with the aid of LiPlaCeT, 4D cell tracking and lineage analysis of populations of dividing and growing cells. The LiPlaCeT plugin contains ad hoc ergonomic curating tools, making it very simple to use for manual cell tracking, especially when the signal-to-noise ratio of images is low or variable in time or 3D space and when automated methods may fail. Performing time-lapse experiments and using cell-tracking data extracted with the assistance of LiPlaCeT, we accomplished deep analyses of cell proliferation and clonal relations in the whole developing LR primordia and constructed genealogical trees. We also used cell-tracking data for endodermis cells of the root apical meristem (RAM) and performed automated analyses of cell population dynamics using ParaView software (also publicly available). Using the RAM as an example, we also showed how LiPlaCeT can be used to generate information at the whole-tissue level regarding cell length, cell position, cell growth rate, cell displacement rate, and proliferation activity. The pipeline will be useful in live-imaging studies of roots and other plant organs to understand complex interactions within proliferating and growing cell populations. The plugin includes a step-by-step user manual and a dataset example that are available at https://www.ibt.unam.mx/documentos/diversos/LiPlaCeT.zip.
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Arabidopsis/fisiología , Proliferación Celular , Rastreo Celular/instrumentación , Células Vegetales/fisiología , Desarrollo de la Planta , Arabidopsis/crecimiento & desarrolloRESUMEN
Despite recent improvements in microscope technologies, segmenting and tracking cells in three-dimensional time-lapse images (3D + T images) to extract their dynamic positions and activities remains a considerable bottleneck in the field. We developed a deep learning-based software pipeline, 3DeeCellTracker, by integrating multiple existing and new techniques including deep learning for tracking. With only one volume of training data, one initial correction, and a few parameter changes, 3DeeCellTracker successfully segmented and tracked ~100 cells in both semi-immobilized and 'straightened' freely moving worm's brain, in a naturally beating zebrafish heart, and ~1000 cells in a 3D cultured tumor spheroid. While these datasets were imaged with highly divergent optical systems, our method tracked 90-100% of the cells in most cases, which is comparable or superior to previous results. These results suggest that 3DeeCellTracker could pave the way for revealing dynamic cell activities in image datasets that have been difficult to analyze.
Microscopes have been used to decrypt the tiny details of life since the 17th century. Now, the advent of 3D microscopy allows scientists to build up detailed pictures of living cells and tissues. In that effort, automation is becoming increasingly important so that scientists can analyze the resulting images and understand how bodies grow, heal and respond to changes such as drug therapies. In particular, algorithms can help to spot cells in the picture (called cell segmentation), and then to follow these cells over time across multiple images (known as cell tracking). However, performing these analyses on 3D images over a given period has been quite challenging. In addition, the algorithms that have already been created are often not user-friendly, and they can only be applied to a specific dataset gathered through a particular scientific method. As a response, Wen et al. developed a new program called 3DeeCellTracker, which runs on a desktop computer and uses a type of artificial intelligence known as deep learning to produce consistent results. Crucially, 3DeeCellTracker can be used to analyze various types of images taken using different types of cutting-edge microscope systems. And indeed, the algorithm was then harnessed to track the activity of nerve cells in moving microscopic worms, of beating heart cells in a young small fish, and of cancer cells grown in the lab. This versatile tool can now be used across biology, medical research and drug development to help monitor cell activities.
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Rastreo Celular/métodos , Aprendizaje Profundo , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Imagen de Lapso de Tiempo/métodos , Animales , Encéfalo/diagnóstico por imagen , Caenorhabditis elegans/citología , Rastreo Celular/instrumentación , Corazón/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/instrumentación , Imagenología Tridimensional/instrumentación , Esferoides Celulares , Imagen de Lapso de Tiempo/instrumentación , Células Tumorales Cultivadas , Pez CebraRESUMEN
Coordinated cell movements drive embryonic development and tissue repair, and can also spread disease. Time-lapse microscopy is an integral part in the study of the cell biology of collective cell movements. Advances in imaging techniques enable monitoring dynamic cellular and molecular events in real time within living animals. Here, we demonstrate the use of spinning disk confocal microscopy to investigate coordinated cell movements and epithelial-to-mesenchymal-like transitions during embryonic wound closure in Drosophila. We describe image-based metrics to quantify the efficiency of collective cell migration. Finally, we show the application of super-resolution radial fluctuation microscopy to obtain multidimensional, super-resolution images of protrusive activity in collectively moving cells in vivo. Together, the methods presented here constitute a toolkit for the modern analysis of collective cell migration in living animals.
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Movimiento Celular , Rastreo Celular/métodos , Embrión no Mamífero/citología , Animales , Rastreo Celular/instrumentación , Drosophila melanogaster , Transición Epitelial-Mesenquimal , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Límite de Detección , Microscopía Confocal/instrumentación , Microscopía Confocal/métodosRESUMEN
In many solid tumors, collective cell invasion prevails over single-cell dissemination strategies. Collective modes of invasion often display specific front/rear cellular organization, where invasive leader cells arise from cancer cell populations or the tumor stroma. Collective invasion involves coordinated cellular movements which require tight mechanical crosstalk through specific combinations of cell-cell interactions and cell-matrix adhesions. Cancer Associated Fibroblasts (CAFs) have been recently reported to drive the dissemination of epithelial cancer cells through ECM remodeling and direct intercellular contact. However, the cooperation between tumor and stromal cells remains poorly understood. Here we present a simple spheroid invasion assay to assess the role of CAFs in the collective migration of epithelial tumor cells. This method enables the characterization of 3D spheroid invasion patterns through live cell fluorescent labeling combined with spinning disc microscopy. When embedded in extracellular matrix, the invasive strands of spheroids can be tracked and leader/follower organization of CAFs and cancer cells can be quantified.
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Fibroblastos Asociados al Cáncer/fisiología , Movimiento Celular , Rastreo Celular/métodos , Imagenología Tridimensional/métodos , Esferoides Celulares/fisiología , Fibroblastos Asociados al Cáncer/citología , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Rastreo Celular/instrumentación , Matriz Extracelular/química , Humanos , Imagenología Tridimensional/instrumentación , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Esferoides Celulares/citología , Células Tumorales CultivadasRESUMEN
The epithelial-mesenchymal transition (EMT) converts coherent epithelial structures into single cells. EMT is a dynamic cellular process that is not systematically completed (not all EMTs lead to single cells) and reversible (cells can re-epithelialize). EMT is orchestrated at multiple levels from transcription, to posttranslational modifications, to protein turnover. It involves remodeling of polarity and adhesion and enhances migratory capabilities. During physiological events such as embryogenesis or wound healing EMT is used to initiate cell migration, but EMT can also occur in pathological settings. In particular, EMT has been linked to fibrosis and cancer. Neural crest (NC) cells, an embryonic stem cell population whose behavior recapitulates the main steps of carcinoma progression, are a great model to study EMT. In this chapter, we provide a fully detailed protocol to extract NC cells from Xenopus embryos and culture them to study the dynamics of cell-cell adhesion, cell motility, and dispersion.
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Rastreo Celular/métodos , Transición Epitelial-Mesenquimal , Cresta Neural/citología , Cultivo Primario de Células/métodos , Animales , Adhesión Celular , Movimiento Celular , Rastreo Celular/instrumentación , XenopusRESUMEN
Nanometric probes based on surface-enhanced Raman scattering (SERS) are promising candidates for all-optical environmental, biological and technological sensing applications with intrinsic quantitative molecular specificity. However, the effectiveness of SERS probes depends on a delicate trade-off between particle size, stability and brightness that has so far hindered their wide application in SERS imaging methodologies. In this Article, we introduce holographic Raman microscopy, which allows single-shot three-dimensional single-particle localization. We validate our approach by simultaneously performing Fourier transform Raman spectroscopy of individual SERS nanoparticles and Raman holography, using shearing interferometry to extract both the phase and the amplitude of wide-field Raman images and ultimately localize and track single SERS nanoparticles inside living cells in three dimensions. Our results represent a step towards multiplexed single-shot three-dimensional concentration mapping in many different scenarios, including live cell and tissue interrogation and complex anti-counterfeiting applications.
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Holografía/instrumentación , Nanopartículas/análisis , Espectrometría Raman/instrumentación , Rastreo Celular/instrumentación , Rastreo Celular/métodos , Diseño de Equipo , Análisis de Fourier , Células HeLa , Holografía/métodos , Humanos , Interferometría/instrumentación , Interferometría/métodos , Nanopartículas/ultraestructura , Tamaño de la Partícula , Espectrometría Raman/métodosRESUMEN
Live cell imaging at high resolution of pollen tubes growing in vitro requires an experimental setup that maintains the elongated cells in a single optical plane and allows for controlled exchange of growth medium. As a low-cost alternative to lithography-based microfluidics, we developed a silicone-based spacer system that allows introducing spatial features and flexible design. These growth chambers can be cleaned and reused repeatedly.
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Rastreo Celular/instrumentación , Tubo Polínico/citología , Arabidopsis , Rastreo Celular/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , SiliconasRESUMEN
Non-invasive tracking of stem cells after transplant is necessary for cell therapy and tissue engineering field. Herein, we introduce natural and biodegradable nanoparticle to develop a highly efficient nanoprobe with the ability to penetrate the stem cell for tracking. Based on the use of (Gd3+) to label stem cells for magnetic resonance imaging (MRI) we synthesized nanoparticle-containing Gd3+. Gd3+ could be used as t1-weighted MRI contrast agents. In this study, chitosan-alginate nanoparticles were synthesized as a clinical Dotarem® carrier for decreased t1-weighted. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier-transform infrared spectroscopy (FTIR) were utilized for nanoprobe characterization and ICP analysis was performed for Gd3+ concentration measurement. The results illustrate that nanoprobes with spherical shape and with a size of 80 nm without any aggregation were obtained. Relaxivity results suggest that r1 in the phantom was 12.8 mM-1s-1 per Gd3+ ion, which is 3.5 times larger than that for Dotarem® (r1 â¼3.6 mM-1s-1 per Gd3+ ion) and this result for synthesized nanoprobe in stem cells 3.56 mM-1s-1 per Gd3+ ion with 2.16 times larger than that for Dotarem® was reported and also enhanced signal in in-vivo imaging was observed. Chitosan-alginate nanoparticles as a novel biocompatible probe for stem cell tracking can be utilized in tissue engineering approach.
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Rastreo Celular/instrumentación , Medios de Contraste/química , Células Madre Mesenquimatosas/citología , Nanopartículas/química , Alginatos/química , Animales , Quitosano/química , Medios de Contraste/metabolismo , Gadolinio/química , Imagen por Resonancia Magnética , Meglumina/química , Células Madre Mesenquimatosas/metabolismo , Ratones , Nanopartículas/metabolismo , Compuestos Organometálicos/química , Tamaño de la PartículaRESUMEN
Rationale: Glioblastoma is the most frequent, primary brain tumor that is characterized by a highly immunosuppressive tumor microenvironment (TME). The TME plays a key role for tumor biology and the effectiveness of immunotherapies. Composition of the TME correlates with overall survival and governs therapy response. Non invasive assessment of the TME has been notoriously difficult. Methods: We have designed an in vivo imaging approach to non invasively visualize innate immune cell dynamics in the TME in a mouse glioma model by correlated MRI and multiphoton microscopy (MR-MPM) using a bimodal, fluorescently labeled iron oxide nanoparticle (NP). The introduction of Teflon cranial windows instead of conventional Titanium rings dramatically reduced susceptibility artifacts on MRI and allowed longitudinal MR-MPM imaging for innate immune cell tracking in the same animal. Results: We visualized tumor associated macrophage and microglia (TAM) dynamics in the TME and dissect the single steps of NP uptake by blood-born monocytes that give rise to tumor-associated macrophages. Next to peripheral NP-loading, we identified a second route of direct nanoparticle uptake via the disrupted blood-brain barrier to directly label tissue resident TAMs. Conclusion: Our approach allows innate immune cell tracking by MRI and multiphoton microscopy in the same animal to longitudinally investigate innate immune cell dynamics in the TME.
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Glioma/diagnóstico por imagen , Inmunidad Innata/inmunología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Microambiente Tumoral/inmunología , Animales , Barrera Hematoencefálica/diagnóstico por imagen , Barrera Hematoencefálica/fisiopatología , Neoplasias Encefálicas/patología , Rastreo Celular/instrumentación , Glioma/patología , Glioma/ultraestructura , Inmunidad Innata/fisiología , Inmunidad Innata/efectos de la radiación , Inmunoterapia/métodos , Macrófagos/inmunología , Nanopartículas Magnéticas de Óxido de Hierro/química , Ratones , Ratones Endogámicos C57BL , Microglía/patología , Nanopartículas/administración & dosificación , Nanopartículas/químicaRESUMEN
While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.
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Anticuerpos Monoclonales/análisis , Rastreo Celular/métodos , Glicoproteínas Hemaglutininas del Virus de la Influenza/análisis , Animales , Rastreo Celular/instrumentación , Femenino , Citometría de Flujo/métodos , Células HEK293 , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Melanoma/química , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Orthomyxoviridae/química , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismoRESUMEN
Mesenchymal stem cell (MSCs) therapy has recently received profound interest as a targeting platform in cancer theranostics because of inherent tumor-homing abilities. However, the terminal tracking of MSCs engraftment by fluorescent in situ hybridization, immuno-histochemistry, and flow-cytometry techniques to translate into clinics is still challenging because of a dearth of inherent MSCs-specific markers and FDA approval for genetic modifications of MSCs. To address this challenge, a cost-effective noninvasive imaging technology based on multifunctional nanocrystals (NCs) with enhanced detection sensitivity, spatial-temporal resolution, and deep-tissue diagnosis is needed to be developed to track the transplanted stem cells. A hassle-free labeling of human umbilical cord Wharton's Jelly (WJ)-derived MSCs with Mn2+ and Gd3+ co-doped CuInS2-ZnS (CIS-ZMGS) NCs has been demonstrated in 2 h without requiring an electroporation process or transfection agents. It has been found that WJ-MSCs labeling did not affect their multilineage differentiation (adipocyte, osteocyte, chondrocyte), immuno-phenotypes (CD44+, CD105+, CD90+), protein (ß-actin, vimentin, CD73, α-SMCA), and gene expressions. Interestingly, CIS-ZMGS-NCs-labeled WJ-MSCs exhibit near-infrared (NIR) fluorescence with a quantum yield of 84%, radiant intensity of â¼3.999 × 1011 (p/s/cm2/sr)/(µW/cm2), magnetic relaxivity (longitudinal r1 = 2.26 mM-1 s-1, transverse r2 = 16.47 mM-1 s-1), and X-ray attenuation (78 HU) potential for early noninvasive multimodality imaging of a subcutaneous melanoma in B16F10-tumor-bearing C57BL/6 mice in 6 h. The ex vivo imaging and inductively coupled plasma mass-spectroscopy analyses of excised organs along with confocal microscopy and immunofluorescence of tumor results also significantly confirmed the positive tropism of CIS-ZMGS-NCs-labeled WJ-MSCs in the tumor environment. Hence, we propose the magnetofluorescent CIS-ZMGS-NCs-labeled WJ-MSCs as a next-generation nanobioprobe of three commonly used imaging modalities for stem cell-assisted anticancer therapy and tracking tissue/organ regenerations.
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Rastreo Celular/métodos , Células Madre Mesenquimatosas/química , Nanopartículas/química , Neoplasias/terapia , Gelatina de Wharton/química , Animales , Rastreo Celular/instrumentación , Fluorescencia , Gadolinio/química , Humanos , Manganeso/química , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Neoplasias/diagnóstico por imagen , Puntos Cuánticos/química , Coloración y Etiquetado , Sulfuros/química , Cordón Umbilical/química , Compuestos de Zinc/químicaRESUMEN
Gold nanoparticles (AuNP) have been extensively developed as contrast agents, theranostic platforms, and probes for molecular imaging. This popularity has yielded a large number of AuNP designs that vary in size, shape, surface functionalization, and assembly, to match very closely the requirements for various imaging applications. Hence, AuNP based probes for molecular imaging allow the use of computed tomography (CT), fluorescence, and other forms of optical imaging, photoacoustic imaging (PAI), and magnetic resonance imaging (MRI), and other newer techniques. The unique physicochemical properties, biocompatibility, and highly developed chemistry of AuNP have facilitated breakthroughs in molecular imaging that allow the detection and imaging of physiological processes with high sensitivity and spatial resolution. In this Review, we summarize the recent advances in molecular imaging achieved using novel AuNP structures, cell tracking using AuNP, targeted AuNP for cancer imaging, and activatable AuNP probes. Finally, the perspectives and current limitations for the clinical translation of AuNP based probes are discussed.
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Oro/análisis , Nanopartículas del Metal/análisis , Imagen Molecular/métodos , Imagen Óptica/métodos , Animales , Rastreo Celular/instrumentación , Rastreo Celular/métodos , Humanos , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/métodos , Imagen Molecular/instrumentación , Imagen Óptica/instrumentación , Técnicas Fotoacústicas/instrumentación , Técnicas Fotoacústicas/métodos , Tomografía Computarizada por Rayos X/instrumentación , Tomografía Computarizada por Rayos X/métodosRESUMEN
Convolutional neural networks (ConvNets) have proven to be successful in both the classification and semantic segmentation of cell images. Here we establish a method for cell type classification utilizing images taken with a benchtop microscope directly from cell culture flasks, eliminating the need for a dedicated imaging platform. Significant flask-to-flask morphological heterogeneity was discovered and overcome to support network generalization to novel data. Cell density was found to be a prominent source of heterogeneity even when cells are not in contact. For the same cell types, expert classification was poor for single-cell images and better for multi-cell images, suggesting experts rely on the identification of characteristic phenotypes within subsets of each population. We also introduce Self-Label Clustering (SLC), an unsupervised clustering method relying on feature extraction from the hidden layers of a ConvNet, capable of cellular morphological phenotyping. This clustering approach is able to identify distinct morphological phenotypes within a cell type, some of which are observed to be cell density dependent. Finally, our cell classification algorithm was able to accurately identify cells in mixed populations, showing that ConvNet cell type classification can be a label-free alternative to traditional cell sorting and identification.
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Rastreo Celular/métodos , Aprendizaje Profundo , Modelos Teóricos , Especificidad de Órganos , Fenotipo , Algoritmos , Rastreo Celular/instrumentación , Microscopía , Redes Neurales de la Computación , Análisis de la Célula Individual/métodosRESUMEN
Intravital imaging is an invaluable tool for studying the expanding range of immune cell functions. Only in vivo can the complex and dynamic behavior of leukocytes and their interactions with their natural microenvironment be observed and quantified. While the capabilities of high-speed, high-resolution confocal and multiphoton microscopes are well-documented and steadily improving, other crucial hardware required for intravital imaging is often developed in-house and less commonly published in detail. In this report, we describe a low-cost, multipurpose, and tissue-stabilizing in vivo imaging platform that enables sensing and regulation of local tissue temperature. The effect of tissue temperature on local blood flow and leukocyte migration is demonstrated in muscle and skin. Two different models of vacuum windows are described in this report, however, the design of the vacuum window can easily be adapted to fit different organs and tissues.
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Rastreo Celular/instrumentación , Rastreo Celular/métodos , Sistema Inmunológico/citología , Microscopía Intravital/instrumentación , Microscopía Intravital/métodos , Temperatura , Animales , Leucocitos/citología , Leucocitos/metabolismo , Masculino , RatonesRESUMEN
We aimed to identify a suitable method for long-term monitoring of the migration and proliferation of mesenchymal stromal cells in stroke models of rats using ferritin transgene expression by magnetic resonance imaging (MRI). Bone marrow mesenchymal stromal cells (BMSCs) were transduced with a lentivirus containing a shuttle plasmid (pCDH-CMV-MCS-EF1-copGFP) carrying the ferritin heavy chain 1 (Fth1) gene. Ferritin expression in stromal cells was evaluated with western blotting and immunofluorescent staining. The iron uptake of Fth1-BMSCs was measured with Prussian blue staining. Following surgical introduction of middle cerebral artery occlusion, Fth1-BMSCs and superparamagnetic iron oxide- (SPIO-) labeled BMSCs were injected through the internal jugular vein. The imaging and signal intensities were monitored by diffusion-weighted imaging (DWI), T2-weighted imaging (T2WI), and susceptibility-weighted imaging (SWI) in vitro and in vivo. Pathology was performed for comparison. We observed that the MRI signal intensity of SPIO-BMSCs gradually reduced over time. Fth1-BMSCs showed the same signal intensity between 10 and 60 days. SWI showed hypointense lesions in the SPIO-BMSC (traceable for 30 d) and Fth1-BMSC groups. T2WI was not sensitive enough to trace Fth1-BMSCs. After transplantation, Prussian blue-stained cells were observed around the infarction area and in the infarction center in both transplantation models. Fth1-BMSCs transplanted for treating focal cerebral infarction were safe, reliable, and traceable by MRI. Fth1 labeling was more stable and suitable than SPIO labeling for long-term tracking. SWI was more sensitive than T2W1 and suitable as the optimal MRI-tracking sequence.
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Rastreo Celular/métodos , Compuestos Férricos/análisis , Ferritinas/análisis , Genes Reporteros , Infarto de la Arteria Cerebral Media/terapia , Imagen por Resonancia Magnética/métodos , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Oxidorreductasas/análisis , Animales , Rastreo Celular/instrumentación , Células Cultivadas , Ferritinas/genética , Ferritinas/metabolismo , Ferritinas/toxicidad , Vectores Genéticos , Infarto de la Arteria Cerebral Media/diagnóstico por imagen , Hierro/metabolismo , Lentivirus/genética , Masculino , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Oxidorreductasas/toxicidad , Prueba de Estudio Conceptual , Biosíntesis de Proteínas , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/análisis , Distribución Tisular , TransgenesRESUMEN
With rapidly advancing microscopy techniques for live cell imaging, we are now able to image groups of migrating cells in many different in vivo contexts. However, as the resulting data sets become larger and more complex, following the behavior of these cells and extracting accurate quantitative data become increasingly challenging. Here we present a protocol for carrying out accurate automated tracking of cells moving over time in 3D, implemented as custom-built macro scripts for ImageJ. As opposed to many generic tracking workflows, the workflow we propose here accounts for the overall movement of the embryo, allows the selection of subgroups of cells, and includes a step for the complete assisted review of all 3D tracks. Furthermore, it is easy to add new custom track measurement to the code provided. Together, these present a reliable method for the precise tracking of cells, from which distinct subsets of cells can be selected from within a population.
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Rastreo Celular/métodos , Imagenología Tridimensional/métodos , Microscopía Intravital/métodos , Imagen de Lapso de Tiempo/métodos , Algoritmos , Animales , Movimiento Celular , Rastreo Celular/instrumentación , Drosophila melanogaster , Embrión no Mamífero/diagnóstico por imagen , Imagenología Tridimensional/instrumentación , Microscopía Intravital/instrumentación , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Reproducibilidad de los Resultados , Programas Informáticos , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
Single-molecule localization microscopy (SMLM) is a rapidly evolving technique to resolve subcellular structures and single-molecule dynamics at the nanoscale. Here, we employ conventional BODIPY conjugates for live-cell SMLM via their previously reported red-shifted ground-state dimers (DII), which transiently form through bi-molecular encounters and emit bright single-molecule fluorescence. We employ the versatility of DII-state SMLM to resolve the nanoscopic spatial regulation and dynamics of single fatty acid analogs (FAas) and lipid droplets (LDs) in living yeast and mammalian cells with two colors. In fed cells, FAas localize to the endoplasmic reticulum and LDs of ~125 nm diameter. Upon fasting, however, FAas form dense, non-LD clusters of ~100 nm diameter at the plasma membrane and transition from free diffusion to confined immobilization. Our reported SMLM capability of conventional BODIPY conjugates is further demonstrated by imaging lysosomes in mammalian cells and enables simple and versatile live-cell imaging of sub-cellular structures at the nanoscale.
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Compuestos de Boro/química , Rastreo Celular/métodos , Colorantes Fluorescentes/química , Imagen Individual de Molécula/métodos , Línea Celular Tumoral , Rastreo Celular/instrumentación , Células/química , Células/citología , Células/metabolismo , Retículo Endoplásmico/química , Retículo Endoplásmico/metabolismo , Humanos , Gotas Lipídicas/química , Gotas Lipídicas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Imagen Individual de Molécula/instrumentaciónRESUMEN
Current approaches for dynamic profiling of single cells rely on dissociated cultures, which lack important biological features existing in tissues. Organotypic slice cultures preserve aspects of structural and synaptic organisation within the brain and are amenable to microscopy, but established techniques are not well adapted for high throughput or longitudinal single cell analysis. Here we developed a custom-built, automated confocal imaging platform, with improved organotypic slice culture and maintenance. The approach enables fully automated image acquisition and four-dimensional tracking of morphological changes within individual cells in organotypic cultures from rodent and human primary tissues for at least 3 weeks. To validate this system, we analysed neurons expressing a disease-associated version of huntingtin (HTT586Q138-EGFP), and observed that they displayed hallmarks of Huntington's disease and died sooner than controls. By facilitating longitudinal single-cell analyses of neuronal physiology, our system bridges scales necessary to attain statistical power to detect developmental and disease phenotypes.
Asunto(s)
Rastreo Celular/métodos , Hipocampo/ultraestructura , Enfermedad de Huntington/patología , Microscopía Confocal/métodos , Neuronas/ultraestructura , Análisis de la Célula Individual/métodos , Sustitución de Aminoácidos , Animales , Animales Recién Nacidos , Diferenciación Celular , Rastreo Celular/instrumentación , Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal/instrumentación , Modelos Biológicos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/ultraestructura , Neuronas/metabolismo , Cultivo Primario de Células , Análisis de la Célula Individual/instrumentación , Técnicas de Cultivo de TejidosRESUMEN
PURPOSE: To develop methods for fluorine-19 (19F) MRI cell tracking in mice on a 3 Tesla clinical scanner. Compared to iron-based cell tracking, 19F MRI has lower sensitivity and, consequently, preclinical 19F cell tracking has only been performed at relatively high magnetic field strengths (> 3 T). Here, we focus on using 19F MRI to detect macrophages in tumors; macrophage density is an indication of tumor aggressiveness and, therefore, 19F MRI could be used as an imaging biomarker. METHODS: Perfluorocarbon (PFC)-labeled macrophages were imaged at 3 T and NMR spectroscopy was performed to validate 19F spin quantification. In vivo 19F MRI was performed on tumor-bearing mice, post-PFC at both 9.4 T and 3 T. 3 T MRI utilized varying NEX and 19F images were analyzed two different ways for 19F quantification. RESULTS: As few as 25,000 cells could be detected as cell pellets at 3 T. 19F quantification in cell pellets by 3 T MRI agreed with NMR spectroscopy. 19F signal was observed in the liver, spleen and tumor in all mice at 9.4 T and 3 T and there was no significant difference in 19F spin quantification. CONCLUSION: This study demonstrates the ability to detect and quantify 19F signal in murine tumors using 19F MRI at 3 T.