Fluorescence images were acquired with a ReScan Confocal instrumentation (RCM 1.1 Visible, Confocal, The Netherlands) equipped with a fixed 50 μm pinhole and combined with a Nikon Eclipse Ti2-A inverted microscope with a motorized Z-stage (DI1500, Nikon). The RCM unit was connected to a Toptica CLE laser with the following excitation modes: 405/488/561/640 nm. Images were taken via an sCMOS camera (PCO edge) using a CFI Plan Apochromat X60 lambda-immersion oil objective (NA 1.4/0.13; Nikon). The instrument was operated by the NIS-Elements software (version 5.11). Images were acquired via a z-stack optical series with a step size of 0.1 microns to cover all structures of interest. To estimate both the total number of cells and the number of binucleated cells present in one cell layer, all images were first segmented using the Otsu thresholding algorithm. Identical brightness and contrast conditions were applied for each data set within one experiment. The total number of cells was obtained using the Fiji plugin “Analyzes particles” with a size of 10 μm (Schindelin et al., 2012 (link)).
Cfi plan apochromat x60 lambda immersion oil objective
Manufactured by Nikon
The CFI Plan Apochromat X60 lambda-immersion oil objective is an optical lens designed for use in microscopy. It has a magnification of 60x and is optimized for use with immersion oil. The objective is part of Nikon's CFI (Crystalline Fluorite) series, which utilizes specialized optical materials and coatings to provide high-quality imaging performance.
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2 protocols using cfi plan apochromat x60 lambda immersion oil objective
1
Quantifying Binucleated Cells in Fluorescence Imaging
2
Automated Image Analysis for Cell Counting
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Fluorescence images were acquired with a ReScan Confocal microscope instrumentation (RCM 1.1 Visible, Confocal.nl) equipped with a fixed 50 µm pinhole size and combined with a Nikon Ti-2 Eclipse microscope equipped with a motorized Z-stage (DI1500, Nikon). The RCM unit was connected to a Toptica CLE laser with the following excitations: 405/488/561/640 nm. Images were taken via an sCMOS camera (PCO edge) using a CFI Plan Apochromat X60 lambda-immersion oil objective (NA 1.4/0.13; Nikon). The instrument was operated by the NIS-Elements software (version 5.11). To calculate the total number of cells and the number of binucleated cells present within one cell layer, all images were first segmented using the Otsu thresholding algorithm. Identical brightness and contrast conditions were applied for each data set within one experiment. The total number of cells was obtained using the Fiji plugin “Analyze particles” applying a size of 10 µm.
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