Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation.

Archana Bhartiya,Stanley W Botchway,Mohammed Yusuf,Ian Robinson

doi:10.3389/fmolb.2021.631774

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.

Highlights

Nuclear chromosomes are composed of chromatin, primarily a complex of deoxyribonucleic acid (DNA) and proteins, which are arranged into elementary structural units of nucleosomes, each made up of 147 base pairs
The nucleosome is composed of eight core histone proteins, in a bead on a string structure forming 11 nm fibers with DNA wrapped around the nucleosomes
The organization of chromatin into these higher-order structures and how they are controlled play a role in the so-called DNA condensation process which still remain a subject of debate as well as representing one of the key challenges in structural biology

Summary

INTRODUCTION

Nuclear chromosomes are composed of chromatin, primarily a complex of deoxyribonucleic acid (DNA) and proteins, which are arranged into elementary structural units of nucleosomes, each made up of 147 base pairs. FLIM (or the excited state lifetime value) is less influenced by molecule concentration and photo-bleaching whilst providing another method of image contrast. Chromosome compaction at nanometer length scales has been suggested along the length of chromosomes imaged using multiphoton FLIM after DAPI staining (Estandarte et al, 2016) alone. This was performed on a classical spread of chromosomes whereby excited state lifetime measurements have been obtained on all 46 chromosomes. Solutions for M-FISH (1) 0.1× Saline-sodium citrate buffer (SSC) stock: Add 1 ml of 20 x SSC (Sigma-Aldrich) in a 200 ml Milli-Q® Millipore water, pH 7.25. In a 50 ml of a 2× SSC, pH 7.45 and keep at room temperature

METHODS

Findings

DATA AVAILABILITY STATEMENT