Approximately 12,000 cells were plated in each well of a 24-well plate (MatTek, Ashland, MA, USA). After 24 h incubation, cells were fixed with 4% para-formaldehyde, permeabilized with Triton X-100 (Sigma-Aldrich), and blocked for nonspecific binding with 5% goat serum. Nuclear DNA was stained with DAPI, and actin was stained with either phalloidin Alexa Fluor 488 (Thermo Fisher Scientific) or Acti-stain 555 (Cytoskeleton Inc., Denver, CO, USA). For each sample, fluorescently labeled cells in seventy-two (8-by-9 square grid) fields of view from a low-magnification lens (10× Plan Fluor lens; N.A. 0.3, Nikon) with a CCD (Hamamatsu OCAR-ER) on a Nikon NI microscope covering a contiguous area of approximately 6.0 mm × 5.0 mm (30.0 mm2) were analyzed. DAPI (nucleus) or phalloidin (F-actin) fluorescence was recorded to obtain morphometric information about the nucleus and cellular body of each cell within the scanning region. Segmentation of nuclear and cellular shape from images was conducted using a custom MATLAB code. At least 500 – 2500 cells/sample were analyzed for statistical analysis.
Plan fluor lens
Manufactured by Nikon
The Plan Fluor lens is a high-quality microscope objective designed for use in laboratory settings. It provides a flat, distortion-free image field and is optimized for fluorescence microscopy applications. The Plan Fluor lens is made with precision optics to deliver accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Triton x 100, by Merck Group (3 mentions)
24 well plate, by MatTek (2 mentions)
Acti stain 555, by Cytoskeleton (2 mentions)
Ocar er, by Nikon (2 mentions)
Digital sight ds qi1mc camera, by Nikon (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Nis element, by Nikon (1 mentions)
Glass bottom 24 well plate, by MatTek (1 mentions)
Ds qimc camera, by Nikon (1 mentions)
Motorized stage, by Prior Scientific (1 mentions)
Eclipse ti e inverted microscope, by Yokogawa (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Nis elements software, by Nikon (1 mentions)
6 protocols using plan fluor lens
1
Fluorescence Microscopy Imaging of Cell Morphology
2
Fluorescence Microscopy Imaging of Cell Morphology
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Approximately 12,000 cells were plated in each well of a 24-well plate (MatTek, Ashland, MA, USA). After 24 h incubation, cells were fixed with 4% para-formaldehyde, permeabilized with Triton X-100 (Sigma-Aldrich), and blocked for nonspecific binding with 5% goat serum. Nuclear DNA was stained with DAPI, and actin was stained with either phalloidin Alexa Fluor 488 (Thermo Fisher Scientific) or Acti-stain 555 (Cytoskeleton Inc., Denver, CO, USA). For each sample, fluorescently labeled cells in seventy-two (8-by-9 square grid) fields of view from a low-magnification lens (10× Plan Fluor lens; N.A. 0.3, Nikon) with a CCD (Hamamatsu OCAR-ER) on a Nikon NI microscope covering a contiguous area of approximately 6.0 mm × 5.0 mm (30.0 mm2) were analyzed. DAPI (nucleus) or phalloidin (F-actin) fluorescence was recorded to obtain morphometric information about the nucleus and cellular body of each cell within the scanning region. Segmentation of nuclear and cellular shape from images was conducted using a custom MATLAB code. At least 500 – 2500 cells/sample were analyzed for statistical analysis.
3
High-Resolution Imaging of Single Cells
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Approximately 12,000 cells were plated in each well of a 24-well glass bottom plate (MatTek, MA), corresponding to approximately 20% surface coverage to ensure single-cell dispersion. After 16 hours of incubation, cells were fixed with 3.7% paraformaldehyde for 12 min at room temperature. Cells were then permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min; nonspecific binding was blocked with PBS supplemented with 1% albumin from bovine serum for 40 min. Nuclear DNA was stained with Hoechst 33342 (Sigma-Aldrich) at 1:50 dilution; F-actin was stained with phalloidin Alexa Fluor 488 (Invitrogen) at a 1:40 dilution. Fluorescently labeled cell samples were visualized with a Nikon digital sight DS-Qi1MC camera mounted on a Nikon TE300 epifluorescence microscope (Nikon Melville, NY) and equipped with a motorized stage and motorized excitation and emission filters (Prior Scientific, Rockland, MA) controlled by NIS-Elements (Nikon). For each sample, 81 (9-by-9 square grid) fields of view from a low-magnification lens (10× Plan Fluor lens; numerical aperture, 0.3; Nikon) were used, which covered a contiguous area of 6.03 mm by 4.73 mm (28.5 mm2). The fluorescence channels for Hoechst 33342 and Alexa Fluor 488 were recorded to obtain the necessary morphometric information about the nucleus and cellular body of each individual cell within the scanning region.
4
Automated Multimodal Imaging of Cells
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All images were collected with a Nikon DS-QiMc camera installed on a customized Nikon TE300 epifluorescent microscope (Nikon, Melville, NY), equipped with a motorized stage and motorized excitation and emission filters (Prior Scientific, Rockland, MA) controlled by Nikon NIS Elements. Images were acquired with a 10× Plan Fluor lens (N.A. 0.3, Nikon, Melville, NY) and different grid numbers with a 20% overlap were acquired in order to ensure that the entire well was imaged. Image size from the camera was 1,280 × 1,280 pixels, and the pixel size 0.57 μm. For immunofluorescence images, three fluorescence channels for Hoechst 33342, Alexa Fluor 488 and Alexa Fluor 647 were recorded, while for AP staining the fluorescence channel Alexa Fluor 568 and phase-contrast channel were recorded.
5
Visualizing Alpha Cell Proliferation
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To visualize the proliferation of alpha, cells were collected and resuspended in 300 μl LPB (containing 4 to 22% sucrose) and placed in the wells of a chambered 8-well μ-slide (ibidi). Cells were imaged on a Nikon Eclipse Ti-E inverted microscope equipped with a confocal spinning disk unit (CSU-X1) operated at 10,000 rpm (Yokogawa), using a 40× Plan Fluor lens (Nikon), and illuminated in bright field. Images were captured every 2 min for 10 to 15 h by an Andor iXon Ultra 897 high-speed electron microscope charge-coupled device (EM-CCD) camera (Andor Technology). Z-stacks were acquired at 0.2- to 0.5-μm intervals using an NI-DAQ-controlled Piezo element. During imaging, wall-less cells were kept at 30°C using an INUG2E-TIZ stage top incubator (Tokai Hit).
6
Visualizing Nuclear Morphology and Actin Cytoskeleton
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GFP–lamin-A was transfected to monitor the nuclear morphology and volume, and EGFP–LifeAct was transfected to visualize actin cytoskeleton that also allows the measurement of cell orientation. The transient transfection complex was prepared in Opti-MEM I reduced serum medium (Gibco, Carlsbad, CA), where FuGENE® HD (Roche, Indianapolis, IN) was mixed as a transfection agent following the manufacturer’s instructions. Confocal microscopy of live cells was conducted using a ×60 Plan Fluor lens (Nikon, N.A. 1.4). Each frame was taken every 4 to 20 min to avoid significant photobleaching during the imaging time of up to 2 h. Z-stacked time-lapsed confocal images were three-dimensionally reconstructed and the 3D rendered images were analyzed using NIS elements software (Nikon).
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