A1r live cell imaging confocal microscope

Manufactured by Nikon

The Nikon A1R Live Cell Imaging Confocal Microscope is a high-performance microscope system designed for advanced live cell imaging. It features a fast, resonant scanner for rapid image acquisition and a highly sensitive detector for low-light conditions. The A1R provides exceptional image quality and resolution, enabling researchers to observe and analyze dynamic cellular processes in real-time.

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Lab products found in correlation

Lsr 2, by BD (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) μ slide vi0, by Ibidi (1 mentions)

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Nikon A1R Live Cell Confocal microscope A1R Live Cell Confocal A1R confocal microscope A1R confocal A1R microscope Confocal microscope

6 protocols using a1r live cell imaging confocal microscope

Endocytic Pathways and Endosomal Escape

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For endocytic pathways study, 2 × 10⁴ cells per well of Hep 3B cells were seeded in 24-well plates and allowed to attach to the bottom of plates for overnight. Then, a cellular uptake assay was carried out by treating cells with enhanced green fluorescent protein mRNA-encapsulated FTT5 LLNs with the addition of different endocytic inhibitors, EIPA, MβCD, and CPZ, respectively. We used BD LSR II flow cytometry analyzer (BD Biosciences, San Jose, CA) for quantifying the cellular uptake of cells in the presence of different inhibitors. For the endosomal escape assay, 2 × 10⁴ cells per well of Hep 3B cells were seeded in an ibidi μ-Dish 35-mm Quad dish (ibidi GmbH, Gräfelfing, Germany) and incubated overnight. Then, cells were treated with Alexa Fluor 647–labeled RNA containing FTT5 LLNs for 6 hours followed by the addition of calcein (150 μg/ml) in each well as previously reported (38 (link)). After washing with phosphate-buffered saline, images of cells were captured by a Nikon A1R Live Cell Imaging Confocal Microscope (Melville, NY) and analyzed by NIS-Elements.

Zhang X., Zhao W., Nguyen G.N., Zhang C., Zeng C., Yan J., Du S., Hou X., Li W., Jiang J., Deng B., McComb D.W., Dorkin R., Shah A., Barrera L., Gregoire F., Singh M., Chen D., Sabatino D.E, & Dong Y. (2020). Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing. Science Advances, 6(34), eabc2315.

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Confocal Imaging of mRNA Delivery in Cell Lines

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Hep3B cells were maintained in Eagle’s Minimum Essential Medium (EMEM, Corning Incorporated, Corning, NY) with 10% FBS, and B16 cells in Dulbecco’s Modified Eagle’s Medium (DMEM, Corning Incorporated, Corning, NY) with 10% FBS. Cells were seeded in Ibidi μ-Dish 35 mm Quad dishes (ibidi GmbH, Gräfelfing, Germany) at the density of 5 × 10⁴ cells/ml in 300 μl medium/well and incubated overnight to allow cell attachment. Then cells were treated with Lipofectamine 3000 (Lipo3000) or TT3-LNPs encapsulating different mRNA probes at the dose of 50ng/well for overnight. After washing with PBS, cells were imaged under a Nikon A1R Live Cell Imaging Confocal Microscope (Melville, NY) and analyzed by NIS-Elements BR imaging software (version 4.20). For colocalization studies, cells were stained with commercialized organelle labelling probes per manufacturers’ instructions before confocal imaging.

Zhao W., Zeng C., Yan J., Du S., Hou X., Zhang C., Li W., Deng B., McComb D.W., Xue Y., Kang D.D, & Dong Y. (2021). Construction of messenger RNA (mRNA) probes delivered by lipid nanoparticles to visualize intracellular protein expression and localization at organelles. Advanced materials (Deerfield Beach, Fla.), 33(45), e2103131.

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Visualizing Pseudomonas Biofilm Formation

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Inoculation of flow cells was done by normalizing overnight cultures to an optical density at 600 nm (OD₆₀₀) of 0.05 and injecting into an Ibidi μ-Slide VI^0.4 (catalog no. 80601; Ibidi). To seed the flow cell surface, the medium flow was suspended, and the bacteria were allowed to adhere at room temperature for 1 h. Flow of 5% (vol/vol) LBNS with 0.1% arabinose was initiated at a rate of 0.15 ml/min and continued for 48 h. Following the biofilm growth period, the flow was terminated, and the biofilms were fixed with 4% paraformaldehyde. Psl was stained with a cocktail of three monoclonal antibodies provided by MedImmune (33 (link)). The antibodies were directly labeled with Alexa Fluor 647 using the Alexa labeling kit (Life Technologies). Confocal images were obtained on a Nikon A1R live-cell imaging confocal microscope. Images were obtained with a 20× oil immersion objective. Images were processed using the FIJI software (45 (link)). Quantitative analyses were performed using the COMSTAT2 software package (46 (link)). Total biomass was determined from Z-stack images using the BIOMASS command with the threshold set at 25. Three independent biofilms were imaged and analyzed. Statistical significance was determined by a one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test (**, P ≤ 0.01; ***, P ≤ 0.001).

Jones C.J, & Wozniak D.J. (2017). Psl Produced by Mucoid Pseudomonas aeruginosa Contributes to the Establishment of Biofilms and Immune Evasion. mBio, 8(3), e00864-17.

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Confocal Imaging of Collagen Fibers

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Collagen fibers within the microfluidic device (Figure 5A) were imaged using confocal microscopy on a Nikon A1R live cell imaging confocal microscope via a 40× 1.3 NA oil immersion lens controlled with the NIS-Elements software. The reflectance signal was detected by exciting samples with a 487 nm laser, passing the reflected light through a 40/60 beam splitter, and collecting it in a detector for visualization of collagen fibers.^{37 (link)} Confocal reflectance stacks of approximately 50 μm in height with a z-step of 0.59 μm were acquired for image analysis (80–90 total images per stack). In each experiment, duplicate devices were imaged for each condition. When imaging, three stacks per device were obtained. Each condition was run three to five times in independent experiments.

Avendano A., Chang J.J., Cortes-Medina M.G., Seibel A.J., Admasu B.R., Boutelle C.M., Bushman A.R., Garg A.A., DeShetler C.M., Cole S.L, & Song J.W. (2020). Integrated Biophysical Characterization of Fibrillar Collagen-Based Hydrogels. ACS biomaterials science & engineering, 6(3), 1408-1417.

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Cellular Uptake and Endosomal Escape Assay

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RAW264.7 cells were seeded in a 24-well plate at 10⁵ cells/well and cultured for 24 h. Then, the cells were treated by FLuc mRNA and Alexa-Fluor 647-labeled RNA (1:1, weight ratio) using Lipofectamine 3000, VcLNPs, or electroporation. After 3 h incubation, cellular uptake was quantified by a flow cytometer (LSRII, BD). To study endocytic pathways of VcLNPs, cellular uptake assay was performed in the presence of different endocytotic inhibitors, 5-(N-methyl-N-isopropyl)amiloride (EIPA), methyl-beta-cyclodextrin (MβCD), and chlorpromazine hydrochloride (CPZ), which inhibit macropinocytosis-, caveolae-, and clathrin-mediated endocytosis, respectively. For the endosomal escape assay, 2 × 10⁴ cells were plated in an imaging dish (Ibidi) for 24 h, and then 150 μg/mL of calcein was added to the cells with or without VcLNPs containing Alexa-Fluor 647-labeled RNA for 6 h at 37 °C^{27 (link)}. After washing with PBS to remove extracellular calcein and VcLNPs, cells were lively imaged under the Nikon A1R Live Cell Imaging Confocal Microscope. The images were analysed by NIS-Elements AR 5.20.00.

Hou X., Zhang X., Zhao W., Zeng C., Deng B., McComb D.W., Du S., Zhang C., Li W, & Dong Y. (2020). Vitamin Lipid Nanoparticles Enable Adoptive Macrophage Transfer for the Treatment of Multidrug-Resistant Bacterial Sepsis. Nature nanotechnology, 15(1), 41-46.

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Lysosomal Localization of eGFP-CatB

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To test whether AMP-CatB can specificly accumulate in the lysosomes, we prepared the eGFP-CatB mRNA and delivered it into RAW264.7 cells using V_CLNPs. Then, the lysosomes were stained with LysoTracker® Red DND-99, a well-established lysosome probe. The co-localization of eGFP-CatB and LysoTracker® Red DND-99 was visualized under the Nikon A1R Live Cell Imaging Confocal Microscope. The images were analysed by NIS-Elements AR 5.20.00.

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