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1.
BMC Biol ; 20(1): 174, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35932043

RESUMO

BACKGROUND: High-throughput live-cell imaging is a powerful tool to study dynamic cellular processes in single cells but creates a bottleneck at the stage of data analysis, due to the large amount of data generated and limitations of analytical pipelines. Recent progress on deep learning dramatically improved cell segmentation and tracking. Nevertheless, manual data validation and correction is typically still required and tools spanning the complete range of image analysis are still needed. RESULTS: We present Cell-ACDC, an open-source user-friendly GUI-based framework written in Python, for segmentation, tracking and cell cycle annotations. We included state-of-the-art deep learning models for single-cell segmentation of mammalian and yeast cells alongside cell tracking methods and an intuitive, semi-automated workflow for cell cycle annotation of single cells. Using Cell-ACDC, we found that mTOR activity in hematopoietic stem cells is largely independent of cell volume. By contrast, smaller cells exhibit higher p38 activity, consistent with a role of p38 in regulation of cell size. Additionally, we show that, in S. cerevisiae, histone Htb1 concentrations decrease with replicative age. CONCLUSIONS: Cell-ACDC provides a framework for the application of state-of-the-art deep learning models to the analysis of live cell imaging data without programming knowledge. Furthermore, it allows for visualization and correction of segmentation and tracking errors as well as annotation of cell cycle stages. We embedded several smart algorithms that make the correction and annotation process fast and intuitive. Finally, the open-source and modularized nature of Cell-ACDC will enable simple and fast integration of new deep learning-based and traditional methods for cell segmentation, tracking, and downstream image analysis. Source code: https://github.com/SchmollerLab/Cell_ACDC.


Assuntos
Processamento de Imagem Assistida por Computador , Saccharomyces cerevisiae , Ciclo Celular , Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Software
2.
PLoS One ; 17(7): e0268297, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35793313

RESUMO

Beyond the more conventional single-cell segmentation and tracking, single-cell cycle dynamics is gaining a growing interest in the field of cell biology. Thanks to sophisticated systems, such as the fluorescent ubiquitination-based cell cycle indicator (FUCCI), it is now possible to study cell proliferation, migration, changes in nuclear morphology and single cell cycle dynamics, quantitatively and in real time. In this work, we introduce FUCCItrack, an all-in-one, semi-automated software to segment, track and visualize FUCCI modified cell lines. A user-friendly complete graphical user interface is presented to record and quantitatively analyze both collective cell proliferation as well as single cell information, including migration and changes in nuclear or cell morphology as a function of cell cycle. To enable full control over the analysis, FUCCItrack also contains features for identification of errors and manual corrections.


Assuntos
Rastreamento de Células , Software , Ciclo Celular , Divisão Celular , Proliferação de Células
3.
Sci Rep ; 12(1): 11899, 2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35831385

RESUMO

VisionTool is an open-source python toolbox for semantic features extraction, capable to provide accurate features detectors for different applications, including motion analysis, markerless pose estimation, face recognition and biological cell tracking. VisionTool leverages transfer-learning with a large variety of deep neural networks allowing high-accuracy features detection with few training data. The toolbox offers a friendly graphical user interface, efficiently guiding the user through the entire process of features extraction. To facilitate broad usage and scientific community contribution, the code and a user guide are available at https://github.com/Malga-Vision/VisionTool.git .


Assuntos
Semântica , Software , Rastreamento de Células
4.
J Biomed Nanotechnol ; 18(4): 1044-1051, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35854460

RESUMO

Mesenchymal stem cells (MSCs) have demonstrated great potential for tissue engineering and regenerative medicine applications. Noninvasive and real-term tracking of transplanted MSCs in vivo is crucial for studying the distribution and migration of MSCs, and their role in tissue injury repair. This study reports on the use of ferrimagnetic vortex iron oxide (FVIO) nanorings modified with anti-human integrin ß1 for specific recognition and magnetic resonance imaging (MRI) tracking of human MSCs (hMSCs). Integrin ß1 is highly expressed at all stem cell proliferation and differentiation stages. Therefore, the anti-integrin ß1 antibody (Ab) introduced in FVIO targets integrin ß1, thus enabling FVIO to target stem cells at any stage. This is unlike the traditional MRI-based monitoring of transplanted stem cells, which usually requires pre-labeling the stem cells with tracers before injection. Because of the ability to recognize hMSCs, the Ab-modified FVIO nanotracers (FVIO-Ab) have the advantage of not requiring pre-labeling before stem cell transplantation. Furthermore, the FVIO-Ab nanotracers have high T*2 contrast resulting from the unique magnetic properties of FVIO which can improve the MRI tracking efficiency of stem cells. This work may provide a new way for stem cell labeling and in vivo MRI tracking, thus reducing the risks associated with stem cell transplantation and promoting clinical translation.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Rastreamento de Células/métodos , Compostos Férricos , Humanos , Integrina beta1 , Imageamento por Ressonância Magnética/métodos
5.
Carbohydr Polym ; 291: 119633, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35698352

RESUMO

Fluorescent non-conjugated nanoparticles without any π-aromatic building blocks are of great interest in biological applications due to their low cytotoxicity and biocompatibility. Herein, the non-conjugated AIE-active polymer nanoparticles bearing ß-cyclodextrin (ß-CD-PNs) were obtained through self-assembly of amphiphilic poly(isobutylene-alt-maleic anhydride)-g-ß-CD (PIMA-g-ß-CD). Unexpectedly, ß-CD-PNs without conventional AIE fluorophores showed strong fluorescence emission in the aggregated state, excellent photostability and water-solubility. More interestingly, ß-CD-PNs showed excellent biocompatibility and low biotoxicity after being co-incubated with HeLa cells and passaged several times. As a result, the strong blue fluorescence signals could still be detected in HeLa cells after up to 15 generations of passages and showed complete cell morphology. Furthermore, ß-CD-PNs could also be used in zebrafish for bioimaging. The results indicated that ß-CD-PNs was a good choice as a tracer for long-term cell tracking and in vivo imaging agent. Our research provided an effective strategy for developing low-toxicity bioprobes based on ß-CD.


Assuntos
Nanopartículas , Polímeros , Animais , Rastreamento de Células , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Iodeto de Potássio , Peixe-Zebra
6.
Stem Cell Res Ther ; 13(1): 266, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729651

RESUMO

BACKGROUND: Endothelial colony forming cells (ECFCs), alone or in combination with mesenchymal stem cells, have been selected as potential therapeutic candidates for critical limb-threatening ischemia (CLTI), mainly for those patients considered as "no-option," due to their capability to enhance revascularization and perfusion recovery of ischemic tissues. Nevertheless, prior to translating cell therapy to the clinic, biodistribution assays are required by regulatory guidelines to ensure biosafety as well as to discard undesired systemic translocations. Different approaches, from imaging technologies to qPCR-based methods, are currently applied. METHODS: In the current study, we have optimized a cell-tracking assay based on DiR fluorescent cell labeling and near-infrared detection for in vivo and ex vivo assays. Briefly, an improved protocol for DiR staining was set up, by incubation of ECFCs with 6.67 µM DiR and intensive washing steps prior cell administration. The minimal signal detected for the residual DiR, remaining after these washes, was considered as a baseline signal to estimate cell amounts correlated to the DiR intensity values registered in vivo. Besides, several assays were also performed to determine any potential effect of DiR over ECFCs functionality. Furthermore, the optimized protocol was applied in combination with qPCR amplification of specific human Alu sequences to assess the final distribution of ECFCs after intramuscular or intravenous administration to a murine model of CLTI. RESULTS: The optimized DiR labeling protocol indicated that ECFCs administered intramuscularly remained mainly within the hind limb muscle while cells injected intravenously were found in the spleen, liver and lungs. CONCLUSION: Overall, the combination of DiR labeling and qPCR analysis in biodistribution assays constitutes a highly sensitive approach to systemically track cells in vivo. Thereby, human ECFCs administered intramuscularly to CLTI mice remained locally within the ischemic tissues, while intravenously injected cells were found in several organs. Our data corroborate the need to perform biodistribution assays in order to define specific parameters such as the optimal delivery route for ECFCs before their application into the clinic.


Assuntos
Rastreamento de Células , Neovascularização Fisiológica , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Isquemia/terapia , Camundongos , Distribuição Tecidual
7.
Chem Rev ; 122(11): 10266-10318, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35549242

RESUMO

The arrival of cell-based therapies is a revolution in medicine. However, its safe clinical application in a rational manner depends on reliable, clinically applicable methods for determining the fate and trafficking of therapeutic cells in vivo using medical imaging techniques─known as in vivo cell tracking. Radionuclide imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) has several advantages over other imaging modalities for cell tracking because of its high sensitivity (requiring low amounts of probe per cell for imaging) and whole-body quantitative imaging capability using clinically available scanners. For cell tracking with radionuclides, ex vivo direct cell radiolabeling, that is, radiolabeling cells before their administration, is the simplest and most robust method, allowing labeling of any cell type without the need for genetic modification. This Review covers the development and application of direct cell radiolabeling probes utilizing a variety of chemical approaches: organic and inorganic/coordination (radio)chemistry, nanomaterials, and biochemistry. We describe the key early developments and the most recent advances in the field, identifying advantages and disadvantages of the different approaches and informing future development and choice of methods for clinical and preclinical application.


Assuntos
Rastreamento de Células , Nanoestruturas , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único
8.
STAR Protoc ; 3(2): 101380, 2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35586316

RESUMO

Cancer cell behavior is highly microenvironment dependent, but we have a limited understanding of malignant cell-microenvironment interactions in vivo. Here, we describe a protocol for xenotransplanting human neuroblastoma (NB) cells into streams of migrating neural crest stem cells in zebrafish embryos, followed by confocal time-lapse imaging and cell tracking. This high-resolution model system facilitates the quantitative spatiotemporal analysis of cancer cell-cell and cell-environment interactions. For complete details on the use and execution of this protocol, please refer to Treffy et al. (2021).


Assuntos
Crista Neural , Neuroblastoma , Animais , Rastreamento de Células , Humanos , Microambiente Tumoral , Peixe-Zebra
9.
PLoS One ; 17(4): e0267740, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35486637

RESUMO

When confined, cells have recently been shown to undergo a phenotypic switch to what has been termed, fast amoeboid (leader bleb-based) migration. However, as this is a nascent area of research, few tools are available for the rapid analysis of cell behavior. Here, we demonstrate that a novel Fiji/ImageJ-based plugin, Analyze_Blebs, can be used to quickly obtain cell migration parameters and morphometrics from time lapse images. As validation, we show that Analyze_Blebs can detect significant differences in cell migration and morphometrics, such as the largest bleb size, upon introducing different live markers of F-actin, including F-tractin and LifeAct tagged with green and red fluorescent proteins. We also demonstrate, using flow cytometry, that live markers increase total levels of F-actin. Furthermore, that F-tractin increases cell stiffness, which was found to correlate with a decrease in migration, thus reaffirming the importance of cell mechanics as a determinant of Leader Bleb-Based Migration (LBBM).


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Rastreamento de Células/métodos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Fenômenos Biológicos
10.
Colloids Surf B Biointerfaces ; 215: 112493, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35430486

RESUMO

Fluorine-19 magnetic resonance imaging (19F MRI) has been a technology of choice for in vivo cell tracking, in which perfluorocarbons (PFCs) nanoemulsions are the most used 19F MRI agents. However, the peculiar physicochemical properties of PFCs may lead to poor cell uptake and misleading cell tracking results. Herein, we employed partially fluorinated aromatic agents to formulate paramagnetic nanoemulsions as novel 19F MRI-fluorescence (FL) dual imaging agents for cell tracking. With the intramolecular π-π interaction, low density and fluorine content, the partially fluorinated agents enable considerable solubilities of functional agents and short relaxation times, which facilitates convenient preparation of stable, biocompatible, and multifunctional nanoemulsions with high 19F MRI sensitivity. Replacing PFCs in 19F MRI nanoemulsions with readily available partially fluorinated aromatic agents may address many issues associated with PFCs and provide a novel strategy for high-performance 19F MRI agents of broad biomedical applications.


Assuntos
Imagem por Ressonância Magnética de Flúor-19 , Fluorcarbonetos , Rastreamento de Células , Corantes Fluorescentes , Fluoretos , Flúor/química , Imagem por Ressonância Magnética de Flúor-19/métodos , Fluorcarbonetos/química , Imageamento por Ressonância Magnética/métodos
11.
J Virol ; 96(7): e0184621, 2022 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-35285687

RESUMO

Real-time imaging of viruses in living cells considerably facilitates the study of virus-host interactions. However, generating a fluorescently labeled recombinant virus is challenging, especially for Zika virus (ZIKV), which causes microcephaly in neonates. The monocistronic nature of the ZIKV genome represents a major challenge for generating a replication-competent genetically engineered ZIKV suitable for real-time imaging. Here, we generated a fluorescent ZIKV by introducing the biarsenical tetracysteine (TC) tag system. After separately inserting the TC tag at six sites in the capsid protein, we found that only when we inserted the TC tag at the site of amino acids 27/28 (AA27/28, or TC27) could the genetically engineered ZIKV be rescued. Importantly, the TC27 ZIKV is characterized as replication and infection competent. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the dynamic nuclear import behavior of the capsid protein. In addition, using the single-particle tracking technology, we acquired real-time imaging evidence that ZIKV moved along the cellular filopodia and entered into the cytoplasm via endocytosis. Thus, we provide a feasible strategy to generate a replication-competent TC-tagged ZIKV for real-time imaging, which should greatly facilitate the study of ZIKV-host interactions in living cells. IMPORTANCE Zika virus (ZIKV) is the mosquito-borne enveloped flavivirus that causes microcephaly in neonates. While real-time imaging plays a critical role in dissecting viral biology, no fluorescent, genetically engineered ZIKV for single-particle tracking is currently available. Here, we generated a replication-competent genetically engineered ZIKV by introducing the tetracysteine (TC) tag into its capsid protein. After labeling the TC tag with the fluorescent biarsenical reagents, we visualized the nuclear import behavior of the capsid protein and the endocytosis process of single ZIKV particle. Taken together, these results demonstrate a fluorescent labeling strategy to track the ZIKV-host interactions at both the protein level and the viral particle level. Our replication-competent TC27 ZIKV should open an avenue to study the ZIKV-host interactions and may provide applications for antiviral screening.


Assuntos
Infecção por Zika virus , Zika virus , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Rastreamento de Células , Humanos , Replicação Viral , Zika virus/genética , Zika virus/metabolismo , Infecção por Zika virus/virologia
12.
Science ; 375(6585): 1182-1187, 2022 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-35271315

RESUMO

Apoptosis of cells and their subsequent removal through efferocytosis occurs in nearly all tissues during development, homeostasis, and disease. However, it has been difficult to track cell death and subsequent corpse removal in vivo. We developed a genetically encoded fluorescent reporter, CharON (Caspase and pH Activated Reporter, Fluorescence ON), that could track emerging apoptotic cells and their efferocytic clearance by phagocytes. Using Drosophila expressing CharON, we uncovered multiple qualitative and quantitative features of coordinated clearance of apoptotic corpses during embryonic development. When confronted with high rates of emerging apoptotic corpses, the macrophages displayed heterogeneity in engulfment behaviors, leading to some efferocytic macrophages carrying high corpse burden. Overburdened macrophages were compromised in clearing wound debris. These findings reveal known and unexpected features of apoptosis and macrophage efferocytosis in vivo.


Assuntos
Apoptose , Rastreamento de Células , Drosophila/embriologia , Desenvolvimento Embrionário , Macrófagos/fisiologia , Fagocitose , Animais , Concentração de Íons de Hidrogênio
13.
Acta Biomater ; 143: 39-51, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35314365

RESUMO

The possibility to produce laser action involving biomaterials, in particular (single) biological cells, has fostered the development of cellular lasers as a novel approach in biophotonics. In this respect, cells that are engineered to carry gain medium (e.g., fluorescent dyes or proteins) are placed inside an optical cavity (i.e., typically a sandwich of highly reflective mirrors), allowing the generation of stimulated emission upon sufficient optical pumping. In another scenario, micron-sized optical resonators supporting whispering-gallery mode (WGM) or semiconductor-based laser probes can be internalized by the cells and support light amplification. This review summarizes the recent advances in the fields of biolasers and cellular lasers, and most importantly, highlights their potential applications in the fields of in vitro and in vivo cell imaging and analysis. They include biosensing (e.g., in vitro detection of sodium chloride (NaCl) concentration), cancer cell imaging, laser-emission-based microscope, cell tracking, cell distinction study, and tissue contraction monitoring in zebrafish. Lastly, several fundamental issues in developing cellular lasers including laser probe fabrication, biocompatibility of the system, and alteration of local refractive index of optical cavities due to protein absorption or probe aggregation are described. Cellular lasers are foreseen as a promising tool to study numerous biological and biophysical phenomena. STATEMENT OF SIGNIFICANCE: Biolasers are generation of laser involving biological materials. Biomaterials, including single cells, can be engineered to incorporate laser probes or fluorescent proteins or fluorophores, and the resulting light emission can be coupled to optical resonator, allowing generation of cellular laser emission upon optical pumping. Unlike fluorescence, this stimulated emission is very sensitive and is capable of detecting small alterations in the optical property of the cells and their environment. In this review, recent development and applications of cellular lasers in the fields of in vitro and in vivo cell imaging, cell tracking, biosensing, and cell/tissue analysis are highlighted. Several challenges in developing cellular lasers including probe fabrication and biocompatibility as well as alteration of cellular environment are explained.


Assuntos
Lasers , Peixe-Zebra , Animais , Materiais Biocompatíveis , Rastreamento de Células , Corantes Fluorescentes
14.
Sci Rep ; 12(1): 2702, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177675

RESUMO

Cell tracking is one of the most critical tools for time-lapse image analysis to observe cell behavior and cell lineages over a long period of time. However, the accompanying graphical user interfaces are often difficult to use and do not incorporate seamless manual correction, data analysis tools, or simple training set design tools if it is machine learning based. In this paper, we introduce our cell tracking software "LIM Tracker". This software has a conventional tracking function consisting of recognition processing and link processing, a sequential search-type tracking function based on pattern matching, and a manual tracking function. LIM Tracker enables the seamless use of these functions. In addition, the system incorporates a highly interactive and interlocking data visualization method, which displays analysis result in real time, making it possible to flexibly correct the data and reduce the burden of tracking work. Moreover, recognition functions with deep learning (DL) are also available, which can be used for a wide range of targets including stain-free images. LIM Tracker allows researchers to track living objects with good usability and high versatility for various targets. We present a tracking case study based on fluorescence microscopy images (NRK-52E/EKAREV-NLS cells or MCF-10A/H2B-iRFP-P2A-mScarlet-I-hGem-P2A-PIP-NLS-mNeonGreen cells) and phase contrast microscopy images (Glioblastoma-astrocytoma U373 cells). LIM Tracker is implemented as a plugin for ImageJ/Fiji. The software can be downloaded from https://github.com/LIMT34/LIM-Tracker .


Assuntos
Rastreamento de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Software , Imagem com Lapso de Tempo/métodos , Animais , Linhagem Celular , Aprendizado Profundo , Humanos , Microscopia de Fluorescência , Ratos
15.
Mol Imaging Biol ; 24(2): 341-351, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35146614

RESUMO

PURPOSE: Reporter gene imaging has been extensively used to longitudinally report on whole-body distribution and viability of transplanted engineered cells. Multi-modal cell tracking can provide complementary information on cell fate. Typical multi-modal reporter gene systems often combine clinical and preclinical modalities. A multi-modal reporter gene system for magnetic resonance imaging (MRI) and positron emission tomography (PET), two clinical modalities, would be advantageous by combining the sensitivity of PET with the high-resolution morphology and non-ionizing nature of MRI. PROCEDURES: We developed and evaluated a dual MRI/PET reporter gene system composed of two human-derived reporter genes that utilize clinical reporter probes for engineered cell detection. As a proof-of-concept, breast cancer cells were engineered to co-express the human organic anion transporter polypeptide 1B3 (OATP1B3) that uptakes the clinical MRI contrast agent gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA), and the human sodium iodide symporter (NIS) which uptakes the PET tracer, [18F] tetrafluoroborate ([18F] TFB). RESULTS: T1-weighted MRI results in mice exhibited significantly higher MRI signals in reporter-gene-engineered mammary fat pad tumors versus contralateral naïve tumors (p < 0.05). No differences in contrast enhancement were observed at 5 h after Gd-EOB-DTPA administration using either intravenous or intraperitoneal injection. We also found significantly higher standard uptake values (SUV) in engineered tumors in comparison to the naïve tumors in [18F]TFB PET images (p < 0.001). Intratumoral heterogeneity in signal enhancement was more conspicuous in relatively higher resolution MR images compared to PET images. CONCLUSIONS: Our study demonstrates the ability to noninvasively track cells engineered with our human-derived dual MRI/PET reporter system, enabling a more comprehensive evaluation of transplanted cells. Future work is focused on applying this tool to track therapeutic cells, which may one day enable the broader application of cell tracking within the healthcare system.


Assuntos
Rastreamento de Células , Gadolínio DTPA , Animais , Meios de Contraste , Genes Reporter , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Tomografia por Emissão de Pósitrons/métodos
16.
Cell Mol Life Sci ; 79(3): 141, 2022 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-35187598

RESUMO

Understanding the generation of complexity in living organisms requires the use of lineage tracing tools at a multicellular scale. In this review, we describe the different multicolor strategies focusing on mouse models expressing several fluorescent reporter proteins, generated by classical (MADM, Brainbow and its multiple derivatives) or acute (StarTrack, CLoNe, MAGIC Markers, iOn, viral vectors) transgenesis. After detailing the multi-reporter genetic strategies that serve as a basis for the establishment of these multicolor mouse models, we briefly mention other animal and cellular models (zebrafish, chicken, drosophila, iPSC) that also rely on these constructs. Then, we highlight practical applications of multicolor mouse models to better understand organogenesis at single progenitor scale (clonal analyses) in the brain and briefly in several other tissues (intestine, skin, vascular, hematopoietic and immune systems). In addition, we detail the critical contribution of multicolor fate mapping strategies in apprehending the fine cellular choreography underlying tissue morphogenesis in several models with a particular focus on brain cytoarchitecture in health and diseases. Finally, we present the latest technological advances in multichannel and in-depth imaging, and automated analyses that enable to better exploit the large amount of data generated from multicolored tissues.


Assuntos
Linhagem da Célula , Rastreamento de Células/métodos , Células Clonais/citologia , Proteínas Luminescentes/metabolismo , Organogênese , Animais , Animais Geneticamente Modificados , Células Clonais/metabolismo , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Especificidade de Órgãos
17.
J Immunol ; 208(6): 1493-1499, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35181636

RESUMO

Two-photon intravital microscopy (2P-IVM) has become a widely used technique to study cell-to-cell interactions in living organisms. Four-dimensional imaging data obtained via 2P-IVM are classically analyzed by performing automated cell tracking, a procedure that computes the trajectories followed by each cell. However, technical artifacts, such as brightness shifts, the presence of autofluorescent objects, and channel crosstalking, affect the specificity of imaging channels for the cells of interest, thus hampering cell detection. Recently, machine learning has been applied to overcome a variety of obstacles in biomedical imaging. However, existing methods are not tailored for the specific problems of intravital imaging of immune cells. Moreover, results are highly dependent on the quality of the annotations provided by the user. In this study, we developed CANCOL, a tool that facilitates the application of machine learning for automated tracking of immune cells in 2P-IVM. CANCOL guides the user during the annotation of specific objects that are problematic for cell tracking when not properly annotated. Then, it computes a virtual colocalization channel that is specific for the cells of interest. We validated the use of CANCOL on challenging 2P-IVM videos from murine organs, obtaining a significant improvement in the accuracy of automated tracking while reducing the time required for manual track curation.


Assuntos
Comunicação Celular , Microscopia Intravital , Animais , Artefatos , Rastreamento de Células , Computadores , Microscopia Intravital/métodos , Camundongos
18.
Sci Rep ; 12(1): 1692, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35105914

RESUMO

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.


Assuntos
Anemia Falciforme/sangue , Rastreamento de Células/instrumentação , Índices de Eritrócitos , Citometria de Fluxo/instrumentação , Microfluídica/instrumentação , Adulto , Estudos de Casos e Controles , Confiabilidade dos Dados , Contagem de Eritrócitos , Eritrócitos , Feminino , Hematócrito , Hemoglobinas/análise , Humanos , Campos Magnéticos , Masculino , Pessoa de Meia-Idade , Valores de Referência , Adulto Jovem
19.
Nat Commun ; 13(1): 713, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132059

RESUMO

Existing approaches to evaluate cell viability involve cell staining with chemical reagents. However, the step of exogenous staining makes these methods undesirable for rapid, nondestructive, and long-term investigation. Here, we present an instantaneous viability assessment of unlabeled cells using phase imaging with computation specificity. This concept utilizes deep learning techniques to compute viability markers associated with the specimen measured by label-free quantitative phase imaging. Demonstrated on different live cell cultures, the proposed method reports approximately 95% accuracy in identifying live and dead cells. The evolution of the cell dry mass and nucleus area for the labeled and unlabeled populations reveal that the chemical reagents decrease viability. The nondestructive approach presented here may find a broad range of applications, from monitoring the production of biopharmaceuticals to assessing the effectiveness of cancer treatments.


Assuntos
Morte Celular/fisiologia , Imagem Óptica/métodos , Animais , Células CHO , Sobrevivência Celular/fisiologia , Rastreamento de Células , Cricetulus , Aprendizado Profundo , Células HeLa , Humanos , Microscopia de Contraste de Fase , Imagem Óptica/instrumentação
20.
Int J Mol Sci ; 23(1)2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-35008993

RESUMO

Cell migration is a complex, tightly regulated multistep process in which cytoskeletal reorganization and focal adhesion redistribution play a central role. Core to both individual and collective migration is the persistent random walk, which is characterized by random force generation and resistance to directional change. We first discuss a model that describes the stochastic movement of ECs and characterizes EC persistence in wound healing. To that end, we pharmacologically disrupted cytoskeletal dynamics, cytochalasin D for actin and nocodazole for tubulin, to understand its contributions to cell morphology, stiffness, and motility. As such, the use of Atomic Force Microscopy (AFM) enabled us to probe the topography and stiffness of ECs, while time lapse microscopy provided observations in wound healing models. Our results suggest that actin and tubulin dynamics contribute to EC shape, compressive moduli, and directional organization in collective migration. Insights from the model and time lapse experiment suggest that EC speed and persistence are directionally organized in wound healing. Pharmacological disruptions suggest that actin and tubulin dynamics play a role in collective migration. Current insights from both the model and experiment represent an important step in understanding the biomechanics of EC migration as a therapeutic target.


Assuntos
Rastreamento de Células , Citoesqueleto/metabolismo , Células Endoteliais/fisiologia , Algoritmos , Animais , Biomarcadores , Movimento Celular , Rastreamento de Células/métodos , Imunofluorescência , Humanos , Microscopia de Força Atômica , Modelos Biológicos , Imagem Molecular/métodos , Cicatrização
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