Document Type
Article
Department
Centre for Regenerative Medicine
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.
Publication (Name of Journal)
Frontiers in Molecular Biosciences
Recommended Citation
Bhartiya, A.,
Robinson, I.,
Yousuf, M.,
Botchway, S. W.
(2021). Combining multicolor FISH with fluorescence lifetime imaging for chromosomal identification and chromosomal sub structure investigation. Frontiers in Molecular Biosciences, 8, 631774.
Available at:
https://ecommons.aku.edu/crm/69
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Comments
Issue, and pagination are not provided by the author/publisher