- 标题
- 摘要
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- 实验方案
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[Methods in Molecular Biology] T-Cell Motility Volume 1930 (Methods and Protocols) || Live Cell Imaging and Analysis to Capture T-Cell Motility in Real-Time
摘要： T-lymphocytes are the principle coordinators of the immune defense system and play a major role in the protection of our body against infections, intruders of non-self, and malignancies. To mount an immune response, T-cells need to be effectively employed to tissue sites of infection or in?ammation and establish contacts with antigen-presenting cells (APCs) or malignant cells. Understanding how T-cells navigate toward their recruitment sites would offer new therapeutic opportunities. Advancement in the hardware and software upgrades of microscopy technology has created several ef?cient and easy-to-operate live cell imaging platforms. In this protocol, we present a generalized and simple-to-follow protocol for live cell imaging of migrating T-cells, which can also be adopted to visualize real-time tracking of intracellular signaling events.
关键词： Advanced microscopy,Cell tracking,Live cell imaging,T-cell migration
更新于2025-09-23 15:23:52
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Automated Intracellular Calcium Profiles Extraction from Endothelial Cells Using Digital Fluorescence Images
摘要： Endothelial cells perform a wide variety of fundamental functions for the cardiovascular system, their proliferation and migration being strongly regulated by their intracellular calcium concentration. Hence it is extremely important to carefully measure endothelial calcium signals under different stimuli. A proposal to automate the intracellular calcium profiles extraction from fluorescence image sequences is presented. Digital image processing techniques were combined with a multi-target tracking approach supported by Kalman estimation. The system was tested with image sequences from two different stimuli. The first one was a chemical stimulus, that is, ATP, which caused small movements in the cells trajectories, thereby suggesting that the bath application of the agonist does not generate significant artifacts. The second one was a mechanical stimulus delivered by a glass microelectrode, which caused major changes in cell trajectories. The importance of the tracking block is evidenced since more accurate profiles were extracted, mainly for cells closest to the stimulated area. Two important contributions of this work are the automatic relocation of the region of interest assigned to the cells and the possibility of data extraction from big image sets in efficient and expedite way. The system may adapt to different kind of cell images and may allow the extraction of other useful features.
关键词： cell tracking,endothelium,Kalman,intracellular calcium,multi-target tracking
更新于2025-09-23 15:21:01
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Fluorescent Nanodiamonds || Cell Tracking and Deep Tissue Imaging
摘要： Cell labeling, both specific and nonspecific, discussed in the previous chapter belongs in part to the field of cell therapy, defined as 'administration of live whole cells or maturation of a specific cell population in a patient for the treatment of a disease'. Cell therapy is an emerging field in the medical research as a promising treatment option for human injuries and diseases. While the first use of cells for therapeutic purposes can be traced to the nineteenth century, much is still lacking in our knowledge of the therapy, including the biodistribution, pharmacokinetics, and pharmacodynamics of transplanted human cells in vivo. Such knowledge is essential for detailed understanding of the cells' development, fate, and contribution to regenerating tissues prior to clinical trials.
关键词： cell tracking,cell therapy,fluorescent nanodiamonds,deep tissue imaging,nanotechnology
更新于2025-09-23 15:21:01
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Dual-Activatable Cell Tracker for Controlled and Prolonged Single-Cell Labeling
摘要： Cell trackers are fluorescent chemical tools that facilitate imaging and tracking cells within live organisms. Despite their versatility, these dyes lack specificity, tend to leak outside of the cell and stain neighboring cells. Here, we report a dual activatable cell tracker for increased spatial and temporal staining control, especially for single-cell tracking. This probe overcomes the typical problems of current cell trackers: off-target staining, high background signal, and leakage from the intracellular medium. Staining with this dye is not cytotoxic and it can be used in sensitive primary cells. Moreover, this dye is resistant to harsh fixation and permeabilization conditions and allows for multi-wavelength studies with confocal microscopy and fluorescence-activated cell sorting. Using this cell tracker, we performed in vivo homing experiments in mice with primary splenocytes and tracked a single cell in a heterogenous, multicellular culture environment for over 20 h. These experiments, in addition to comparative proliferation studies with other cell trackers, demonstrated that the signal from this dye is retained in cells for over 72 h after photoactivation. We envision that this type of probes will facilitate the analysis of single-cell behavior and migration in cell culture and in vivo experiments.
关键词： fluorescent dyes,photoactivation,single-cell tracking,cell trackers,intracellular labeling
更新于2025-09-23 15:19:57
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Effects of freezing on mesenchymal stem cells labeled with gold nanoparticles
摘要： Stem cell therapies are a promising treatment for many patients suffering from diseases with poor prognosis. However, clinical translation is inhibited by a lack of in vivo monitoring techniques to track stem cells throughout the course of treatment. Ultrasound-guided photoacoustic imaging of nanoparticle-labeled stem cells may be a solution. To allow photoacoustic tracking, stem cells must be labeled with an optically absorbing contrast agent. Gold nanoparticles are one option due to their cytocompatibility and strong optical absorption in the near-infrared region. However, stem cell labeling can require up to 24-hour incubation with nanoparticles in culture prior to use. Although stem cell monitoring is critically needed, the additional preparation time may not be feasible – it is cost prohibitive and stem cell treatments should be readily available in emergency situations as well as scheduled procedures. To remedy this, stem cells can be labeled prior to freezing and long-term storage. While it is well known that stem cells retain their cellular function after freezing, storage, and thawing, the impact of gold nanoparticles on this process has yet to be investigated. Therefore, we assessed the viability, multipotency, and photoacoustic activity of gold nanosphere-labeled mesenchymal stem cells after freezing, storage, and thawing for one week, one month, or two months and compared to unlabeled, na?ve mesenchymal stem cells which were frozen, stored, and thawed at the same time points. Results indicated no substantial change in viability as assessed by the MTT assay. Differentiation, observed through adipogenesis and osteogenesis, was also comparable to controls. Lastly, strong photoacoustic signals and similar photoacoustic spectral signatures remained. Further studies involving more diverse stem cell types and nanoparticles are required, but our data suggests that function and imaging properties of nanoparticle-labeled stem cells are maintained after freezing and storage, which improves translation of stem cell monitoring techniques by simplifying integration with clinical protocols.
关键词： photoacoustic imaging,cell tracking,freezing,Gold nanospheres,ultrasound,stem cells
更新于2025-09-16 10:30:52
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Dual-Modal Imaging-Guided Precise Tracking of Bioorthogonally Labeled Mesenchymal Stem Cells in Mouse Brain Stroke
摘要： Non-invasive and precise stem cell tracking after transplantation in living subject is very important to monitor both stem cell destinations and their in vivo fate, which was closely related to their therapeutic efficacy. Herein, we developed bicyclo[6.1.0]nonyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-NPs) as a delivery system of dual-modal stem cell imaging probes. Near-infrared fluorescent (NIRF) dye, Cy5.5, was chemically conjugated to the BCN-NPs and then oleic acid-coated superparamagnetic iron oxide nanoparticles (OA-Fe3O4 NPs) were encapsulated into BCN-NPs, in resulting Cy5.5-labeled and OA-Fe3O4 NP-encapsulated BCN-NPs (BCN-dual-NPs). For bioorthogonal labeling of human adipose-derived mesenchymal stem cells (hMSCs), firstly, hMSCs were treated with tetra-acetylated N-azidoacetyl-D-mannosamine (Ac4ManNAz) for generating azide (-N3) groups onto their surface via metabolic glycoengineering. Second, azide groups on the cell surface were successfully chemically labeled with BCN-dual-NPs via bioorthogonal click chemistry in vitro. This bioorthogonal labeling of hMSCs could greatly increase the cell labeling efficiency, safety, and imaging sensitivity, compared to only nanoparticle-derived labeling technology. The dual-modal imaging-guided precise tracking of bioorthogonally labeled hMSCs was tested in the photothrombotic stroke mouse model via intraparenchymal injection. Finally, BCN-dual-NPs-labeled hMSCs could be effectively tracked of their migration from implanted site to brain stroke lesion using NIRF/T2-weighted magnetic resonance (MR) dual-modal imaging for 14 days. Our observation would provide a potential application of bioorthogonally labeled stem cell imaging in regenerative medicine by providing safety and high labeling efficiency in vitro and in vivo.
关键词： metabolic engineering,dual-modal imaging,bioorthogonal click chemistry,stem cell tracking,imaging probe,brain stroke
更新于2025-09-16 10:30:52
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Non-invasive imaging reveals conditions that impact distribution and persistence of cells after in vivo administration
摘要： Background: Cell-based regenerative medicine therapies are now frequently tested in clinical trials. In many conditions, cell therapies are administered systemically, but there is little understanding of their fate, and adverse events are often under-reported. Currently, it is only possible to assess safety and fate of cell therapies in preclinical studies, specifically by monitoring animals longitudinally using multi-modal imaging approaches. Here, using a suite of in vivo imaging modalities to explore the fate of a range of human and murine cells, we investigate how route of administration, cell type and host immune status affect the fate of administered cells. Methods: We applied a unique imaging platform combining bioluminescence, optoacoustic and magnetic resonance imaging modalities to assess the safety of different human and murine cell types by following their biodistribution and persistence in mice following administration into the venous or arterial system. Results: Longitudinal imaging analyses (i) suggested that the intra-arterial route may be more hazardous than intravenous administration for certain cell types, (ii) revealed that the potential of a mouse mesenchymal stem/stromal cell (MSC) line to form tumours depended on administration route and mouse strain and (iii) indicated that clinically tested human umbilical cord (hUC)-derived MSCs can transiently and unexpectedly proliferate when administered intravenously to mice. Conclusions: In order to perform an adequate safety assessment of potential cell-based therapies, a thorough understanding of cell biodistribution and fate post administration is required. The non-invasive imaging platform used here can expose not only the general organ distribution of these therapies, but also a detailed view of their presence within different organs and, importantly, tumourigenic potential. Our observation that the hUC-MSCs but not the human bone marrow (hBM)-derived MSCs persisted for a period in some animals suggests that therapies with these cells should proceed with caution.
关键词： Safety,Mesenchymal stem/stromal cells,Preclinical models,Multi-modal imaging,Cell therapies,Cell tracking
更新于2025-09-11 14:15:04
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Centroid tracking and velocity measurement of white blood cell in video
摘要： Automated blood cells tracking system has a vital role as the tracking process reflects the blood cell characteristics and indicates several diseases. Blood cells tracking is challenging due to the non-rigid shapes of the blood cells, and the variability in their videos along with the existence of different moving objects in the blood. To tackle such challenges, we proposed a green star based centroid (GSBC) moving white blood cell (WBC) tracking algorithm to measure its velocity and draw its trajectory. The proposed cell tracking system consists of two stages, namely WBC detection and blob analysis, and fine tuning the tracking process by determine the centroid of the WBC, and mark the centroid for further fine tracking and to exclude the bacteria from the bounding box. Furthermore, the speed and the trajectory of the WBC motion are recorded and plotted. In the experiments, an optical flow technique is compared with the proposed tracking system showing the superiority of the proposed system as the optical flow method failed to track the WBC. The proposed system identified the WBC accurately, while the optical flow identified all other objects lead to its disability to track the WBC.
关键词： Trajectory analysis,Video processing,Cell detection,Velocity measurements,Blob analysis,White blood cell,Tracking system
更新于2025-09-10 09:29:36
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Indocyanine Green labeling for optical and photoacoustic imaging of Mesenchymal Stem Cells after in vivo transplantation
摘要： The transplantation of Mesenchymal Stem Cells (MSCs) holds great promise for the treatment of a plethora of human diseases, but new non-invasive procedures are needed to monitor the cell fate in vivo. Already largely used in medical diagnostics, the fluorescent dye Indocyanine Green (ICG) is an established dye to track limited numbers of cells by optical imaging, but it can be visualized also by Photoacoustic Imaging (PAI), which provides a higher spatial resolution than pure near infrared fluorescence imaging (NIRF). Because of its successful use in clinical and preclinical examinations, we chose ICG as PAI cell labeling agent. Optimal incubation conditions were defined for an efficient and clinically translatable MSC labeling protocol, such that no cytotoxicity or alterations of the phenotypic profile were observed, and a consistent intracellular uptake of the molecule was achieved. Suspensions of ICG-labeled cells were both optically and optoacoustically detected in vitro, revealing a certain variability in the photoacoustic spectra acquired by varying the excitation wavelength from 680 to 970 nm. Intramuscular engraftments of ICG-labeled MSCs were clearly visualized by both PAI and NIRF over few days after transplantation in the hindlimb of healthy mice, suggesting that the proposed technique retains a considerable potential in the field of transplantation-focused research and therapy.
关键词： Indocyanine Green,Near Infrared Fluorescence Imaging,Photoacoustic Imaging,Cell tracking,Stem Cells
更新于2025-09-10 09:29:36