Bhartiya, A;
Robinson, I;
Yusuf, M;
Botchway, SW;
(2021)
Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation.
Frontiers in Molecular Biosciences
, 8
, Article 631774. 10.3389/fmolb.2021.631774.
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Abstract
Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes.
Type: | Article |
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Title: | Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation |
Open access status: | An open access version is available from UCL Discovery |
DOI: | 10.3389/fmolb.2021.631774 |
Publisher version: | https://doi.org/10.3389/fmolb.2021.631774 |
Language: | English |
Additional information: | © 2021 Bhartiya, Robinson, Yusuf and Botchway. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
Keywords: | Chromosome, fluorescence lifetime imaging, multicolor FISH, microscopy, multiphoton, karyotyping |
UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > London Centre for Nanotechnology |
URI: | https://discovery.ucl.ac.uk/id/eprint/10125755 |
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