Aug 22, 2025

4D ALIGNMENT ANALYSIS PROTOCOL V.2

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvodrmzg4o/v2
  • Yuanzhuo Zhou1,2,
  • Kohei Asai1,2,
  • Hirohisa Kyogoku1,3,
  • Tomoya S. Kitajima1,2
  • 1Laboratory for Chromosome Segregation, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan;
  • 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan;
  • 3Graduate School of Agricultural Science, Kobe University, Kobe, Japan
  • Designing protein-based artificial kinetochore
Yuanzhuo Zhou
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvodrmzg4o/v2
Protocol Citation: Yuanzhuo Zhou, Kohei Asai, Hirohisa Kyogoku, Tomoya S. Kitajima 2025. 4D ALIGNMENT ANALYSIS PROTOCOL. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvodrmzg4o/v2Version created by Yuanzhuo Zhou
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 21, 2025
Last Modified: August 22, 2025
Protocol  Integer ID: 225235
Keywords: alignment of chromosome, 4d analysis, alignment, chromosome, gem foci, imaris software, 4d, using imaris software, analysis protocol, spindle over time, spindle, analysis protocol this protocol
Abstract
This protocol describes a 4D analysis workflow for reconstructing and quantifying the alignment of chromosomes and NDC80-GEM foci in the spindle over time using Imaris software.
Guidelines
1. Open time-lapse image datasets in Imaris software (Bitplane).
2. Reconstruct and segment chromosomes and NDC80-GEM foci in 3D using the automatic thresholding function.
3. Manually validate all segmented surfaces according to fluorescent signals and record it.
4. Manually estimate the spindle axis at each timepoint based on chromosome distribution and record it using the camera manager function.
5. Define the spindle center as the center of mass of the chromosomes.
6. For timepoints before the spindle axis is established (e.g., 2 hours after NEBD), apply the spindle axis determined at 3 hours after NEBD.
7. In the alignment analysis, manually exclude NDC80-GEM clusters located outside the spindle.
Protocol
Open time-lapse image datasets in Imaris software (Bitplane).
Reconstruct and segment chromosomes and NDC80-GEM foci in 3D using the automatic thresholding function.
Manually validate all segmented surfaces according to fluorescent signals and record it.
Manually estimate the spindle axis at each timepoint based on chromosome distribution and record it using the camera manager function.
Define the spindle center as the center of mass of the chromosomes.
For timepoints before the spindle axis is established (e.g., 2 hours after NEBD), apply the spindle axis determined at 3 hours after NEBD.
In the alignment analysis, manually exclude NDC80-GEM clusters located outside the spindle.