Wide-field images were acquired on a Nikon Eclipse Ni-E upright microscope equipped with a CFI Plan Apochromat Lambda 20× 0.75 NA objective lens (Nikon), Nikon motorized XY stage, and Hamamatsu ORCA-Flash4.0 LT scientific CMOS camera, driven by NIS-Elements. EB1-GFP was supplemented to the reactions at a final concentration of 200–400 nM. EB1 comets were imaged at 2 s intervals, and analyzed with TrackMate (Tinevez et al., 2017 (link)). For identifying spots, the LoG detector was used with 2.0 µm spot diameter, threshold 10.0, no median filter, subpixel localization enabled. For linking tracks, the LAP tracker was applied with a max search radius of 3 µm without gap closing. The results were further analyzed by a custom script written in Python to calculate the polymerization velocity (average of the frame-to-frame velocity for each track) and the catastrophe rate (fitting an exponential function to the comet duration distribution). The DABEST-Python package was used for statistics and effect size estimation (Ho et al., 2019 (link)).
Cfi plan apochromat lambda 20 0.75 na objective lens
Manufactured by Nikon
The CFI Plan Apochromat Lambda 20× 0.75 NA objective lens is a high-performance microscope objective designed for use in various laboratory applications. It offers a 20× magnification and a numerical aperture of 0.75, which enables it to capture detailed images with high resolution and contrast. The lens is part of Nikon's CFI (Chromatic-aberration-Free Infinity) optical system, ensuring superior optical performance and color correction.
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4 protocols using cfi plan apochromat lambda 20 0.75 na objective lens
1
EB1 Comet Dynamics Analysis
2
Quantitative Analysis of Microtubule Dynamics
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Wide-field images were acquired on a Nikon Eclipse Ni-E upright microscope equipped with a CFI Plan Apochromat Lambda 20× 0.75 NA objective lens (Nikon), Nikon motorized XY stage, and Andor Zyla 4.2 Plus scientific CMOS camera, driven by NIS-Elements. Alexa 647–labeled bovine tubulin, EB1-GFP (110 nM final), Tau-mCherry (20 nM final; Mooney et al., 2017 (link)), and/or Alexa 568–labeled 10 kDa dextran were supplemented to the reactions. Dark current subtraction and flat field correction were applied to both channels. A custom script written in MATLAB was used to quantify the fluorescent intensity as a function of radial distance from the MTOC (Pelletier et al., 2020 ). Alexa 568–labeled dextran showed a flat intensity profile with respect to the aster as expected for a molecule that shows no spatial variation (Supplemental Figure 4). Spot detection of EB1 comets was performed with TrackMate as described above.
3
Microscopic Tubulin and EB1 Analysis
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Wide-field images were acquired on a Nikon Eclipse Ni-E upright microscope equipped with a CFI Plan Apochromat Lambda 20× 0.75 N.A. objective lens (Nikon), Nikon motorized XY stage, and Andor Zyla 4.2 Plus scientific CMOS camera, driven by NIS-Elements. Alexa647-labelled bovine tubulin and EB1-GFP were supplemented to the reactions. Dark current subtraction and flat field correction were applied to both channels. A custom script written in Matlab was used to quantify the fluorescent intensity as a function of radial distance from the MTOC (Pelletier et al. 2020) . Spot detection of EB1 comets was performed with TrackMate as described above.
4
Live-cell EB1 comet dynamics analysis
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Wide-field images were acquired on a Nikon Eclipse Ni-E upright microscope equipped with a CFI Plan Apochromat Lambda 20× 0.75 N.A. objective lens (Nikon), Nikon motorized XY stage, and Hamamatsu ORCA-Flash4.0 LT scientific CMOS camera, driven by NIS-Elements. EB1-GFP was supplemented to the reactions at a final concentration of 200-400 nM. EB1 comets were imaged at 2 second intervals, and analyzed with TrackMate (Tinevez et al. 2017) . For identifying spots, the LoG detector was used with with 2.0 micron spot diameter, threshold 10.0, no median filter, sub-pixel localization enabled. For linking tracks, the LAP tracker was applied with max search radius of 3 micron without gap closing. The results were further analyzed by a custom script written in Python to calculate the polymerization velocity (average of the frame-toframe velocity for each track) and the catastrophe rate (fitting an exponential function to the comet duration distribution). The DABEST-Python package was used for statistics and effect size estimation (Ho et al. 2019) .
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