Quantification of GFP Signals by Fluorescent Microscopy and Flow Cytometry
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Fluorescent labelling
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The limitations of optical microscopy to determine the cellular localization of label-free nanoparticles prevent a solid prediction of the cellular effect of particles intended for medical applications. To avoid the strong physicochemical changes associated with fluorescent labelling, which often result in differences in cellular uptake, efficiency and toxicity of particles, novel detection techniques are required.In the present study, we determined the intracellular content of unlabeled SPIONs by analyzing refractive index (RI)-based images from holotomographic three-dimensional (3D) microscopy and side scatter data measured by flow cytometry. The results were compared with the actual cellular SPION amount as quantified by atomic emission spectroscopy (AES).Live cell imaging by 3D holotomographic microscopy demonstrated cell-specific differences in intracellular nanoparticle uptake in different pancreatic cell lines. Thus, treatment of PANC-1SMAD4 (1-4) and PANC-1SMAD4 (2-6) with SPIONs resulted in a significant increase in number of areas with higher RI, whereas in PANC-1, SUIT-2 and PaCa DD183, only a minimal increase of spots with high RI was observed. The increase in areas with high RI was in accordance with the SPION content determined by quantitative iron measurements using AES. In contrast, determination of the SPION amount by flow cytometry was strongly cell type-dependent and did not allow the discrimination between intracellular and membrane-bound SPIONs. However, flow cytometry is a very rapid and reliable method to assess the cellular toxicity and allows an estimation of the cell-associated SPION content.Holotomographic 3D microscopy is a useful method to distinguish between intracellular and membrane-associated particles. Thus, it provides a valuable tool for scientists to evaluate the cellular localization and the particle load, which facilitates prediction of potential toxicity and efficiency of nanoparticles for medical applications.
Iron oxide nanoparticles
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Laser Scanning
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The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969-973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one-photon confocal and by two-photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.
Two-photon excitation microscopy
Fluorescence-lifetime imaging microscopy
Fluorescent protein
Protein tag
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Fluorescent labelling
Cytometry
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Abstract Toxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronically infected animals and humans. Cysts can reactivate leading to severe pathologies. They also contribute to the transmission of Toxoplasma infection in humans by ingestion of undercooked meat. Classically, the quantification of cyst burden in tissues uses microscopy methods, which are laborious and time consuming. Here, we have developed automated protocols to quantify cysts, based on flow cytometry or high‐throughput microscopy. Brains of rodents infected with cysts of Prugniaud strain were incubated with the FITC‐ Dolichos biflorus lectin and analyzed by flow cytometry and high‐throughput epifluorescence microscopy. The comparison of cyst counts by manual epifluorescence microscopy to flow cytometry or to high‐throughput epifluorescence microscopy revealed a good correlation ( r = 0.934, r = 0.993, P < 0.001 respectively). High‐throughput epifluorescence microscopy was found to be more specific and sensitive than flow cytometry and easier to use for large series of samples. This reliable and easy protocol allow the specific detection of Toxoplasma cysts in brain, even at low concentrations; it could be a new way to detect them in water and in contaminate food. © 2011 International Society for Advancement of Cytometry
Cytometry
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We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.
Fluorescent labelling
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Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.
Fluorescent staining
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Fluorescent protein
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Abstract Molecular cloning offers an opportunity for designing constructs for expressing chimeric proteins containing incorporated reporter molecules. In this approach, molecules are identified based upon reporter molecules which are expressed within cells as a result of transfection, instead of marking them through immunolabeling, in situ hybridization, or derivative incorporation. This approach is particularly useful for studies involving integrated microscopy. Integrated microscopy allows us to assemble images of the same cell obtained with different microscopes into one comprehensive message concerning cellular functions. Integration of fluorescence and electron spectroscopic imaging is particularly promising. The main advantage of this approach relies in overcoming limitations of each type of microscopy alone i.e. in studies on living cells limitations in spatial resolution of light microscopy and in analysis of supramolecular organization, limitation of electron microscopy to frozen or fixed cells. For this purpose, special reporter molecules suitable for selected modes of microscopy have to be used.
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Flow cytometry and microscopy are equally important in cell analysis. However, few reports have compared the optical data (cell size, internal complexity and fluorescent signal) from flow cytometry and microscopy. In this study, we compared the scattergram from XN-series, a flow cytometry based hematology analyzer with microscopic images of similarly treated leukocytes, and investigated the correlation between the appearance in the scattergram and cell size, internal complexity and fluorescence intensity. Healthy human peripheral blood was analyzed using the XN analyzer. For microscopic comparison, five types of leukocytes (monocytes, lymphocytes, basophils, neutrophils and eosinophils) were isolated from the peripheral blood by centrifugation and magnetic cell sorting, treated with a specific reagent and analyzed using electron microscopy, laser microscopy and confocal laser microscopy. Cell size, residual internal structures and fluorescence intensity correlated with intensity of forward-scattering, side scattering and fluorescent light. In this study, optical data from a clinically used hematology analyzer was clarified using microscopic images.
Cytometry
Cell Sorting
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