Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells

Tsvetelina E Germanova; Emanuele Roscioli; Jonathan U Harrison; Andrew D McAinsh; Nigel J Burroughs

doi:10.1016/j.xpro.2021.100774

Subcellular Euclidean distance measurements with multicolor fluorescence localization imaging in cultured cells

STAR Protoc. 2021 Nov 15;2(4):100774. doi: 10.1016/j.xpro.2021.100774. eCollection 2021 Dec 17.

Authors

Tsvetelina E Germanova^{1

2}, Emanuele Roscioli^{1

2}, Jonathan U Harrison³, Andrew D McAinsh^{1

2}, Nigel J Burroughs^{1

3}

Affiliations

¹ Centre for Mechanochemical Cell Biology, University of Warwick, Coventry CV47AL, UK.
² Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.
³ Mathematics Institute, University of Warwick, Coventry CV4 7AL, UK.

Abstract

This protocol measures the 3D Euclidean distance (Δ_3D) between two/three fluorescently labeled kinetochore components in fixed samples using Kinetochore Delta software (KiDv1.0.1, MATLAB based). Overestimation of mean Δ_3D is corrected through a Bayesian algorithm, with Δ_EC distances reflecting the ensemble average positions of fluorophores within a kinetochore population. This package also enables kinetochore categorization, which can be used to sub-sample kinetochores and measure Δ_EC. Together, this allows the dynamic architecture of human kinetochores to be investigated (tested in hTERT-RPE1 cells). For complete details on the use and execution of this protocol, please refer to Roscioli et al. (2020).

Keywords: Cell Biology; Cell culture; Microscopy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Algorithms
Cells, Cultured
Fluorescent Dyes / chemistry
Humans
Image Processing, Computer-Assisted / methods*
Intracellular Space / physiology*
Kinetochores / physiology
Microscopy, Fluorescence / methods*
Software

Substances

Fluorescent Dyes

Abstract

Publication types

MeSH terms

Substances

Grants and funding