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

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.

Ultra-Structural Imaging Provides 3D Organization of 46 Chromosomes of a Human Lymphocyte Prophase Nucleus

“…Multicolor fluorescence in situ hybridization (mFISH) was performed using a 24Xcyte mFISH probe kit according to the manufacturer’s instructions (MetaSystems, Altlussheim Germany) as well as previously published protocols [ 52 , 65 , 66 ]. Briefly, slides were washed in 100% ethanol for 1 min and then left to air dry.…”

Light Sheet Microscopy and 3D Analysis of Human iPSC-derived Embryoid Bodies

“…M-FISH was performed using a 24Xcyte mFISH probe kit (MetaSystems, Germany) according to manufacturer's (MetaSystems, Germany, http://www.metasystems-international.com) and previously published protocols [37,47,48,49]. Briefly, slides were washed in an ethanol series 70%, 85% and 100% ethanol for 1 minute each and then left to air dry.…”