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Measuring Nanoscale Chromatin Heterogeneity with Partial Wave Spectroscopic Microscopy

  • Scott Gladstein
  • Andrew Stawarz
  • Luay M. Almassalha
  • Lusik Cherkezyan
  • John E. Chandler
  • Xiang Zhou
  • Hariharan Subramanian
  • Vadim Backman
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1745)

Abstract

Despite extensive research in the area, current understanding of the structural organization of higher-order chromatin topology (between 20 and 200 nm) is limited due to a lack of proper imaging techniques at these length scales. The organization of chromatin at these scales defines the physical context (nanoenvironment) in which many important biological processes occur. Improving our understanding of the nanoenvironment is crucial because it has been shown to play a critical functional role in the regulation of chemical reactions. Recent progress in partial wave spectroscopic (PWS) microscopy enables real-time measurement of higher-order chromatin organization within label-free live cells. Specifically, PWS quantifies the nanoscale variations in mass density (heterogeneity) within the cell. These advancements have made it possible to study the functional role of chromatin topology, such as its regulation of the global transcriptional state of the cell and its role in the development of cancer. In this chapter, the importance of studying chromatin topology is explained, the theory and instrumentation of PWS are described, the measurements and analysis processes for PWS are laid out in detail, and common issues, troubleshooting steps, and validation techniques are provided.

Keywords

Partial wave spectroscopic microscopy Chromatin structure Chromatin topology Nanoscale imaging Fractal dimension Cancer development Heterogeneity Live cell imaging Transcriptional 

Supplementary material

Video 1

This video shows the dynamic nature of chromatin structure by continuously imaging live MDA-MB-231 cells over the course of 30 min (AVI 76806 kb)

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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Scott Gladstein
    • 1
  • Andrew Stawarz
    • 1
  • Luay M. Almassalha
    • 1
  • Lusik Cherkezyan
    • 1
  • John E. Chandler
    • 1
  • Xiang Zhou
    • 1
  • Hariharan Subramanian
    • 1
  • Vadim Backman
    • 1
  1. 1.Department of Biomedical EngineeringNorthwestern UniversityEvanstonUSA

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