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.
Ocar er
Manufactured by Nikon
The OCAR-ER is a compact, high-performance optical camera adapter designed for laboratory and research applications. The device allows for the integration of digital cameras with a wide range of optical instruments, enabling seamless image capture and data acquisition. Its core function is to provide a stable and reliable interface between the camera and the optical system, ensuring accurate data collection and analysis.
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2 protocols using ocar er
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.
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