Community Research and Development Information Service - CORDIS

H2020

VCSD — Result In Brief

Project ID: 656873
Funded under: H2020-EU.1.3.2.
Country: Spain
Domain: Fundamental Research

Chromatin seen at the molecular level

Fluorescence microscopy is an essential tool to visualise cell biology in situ, but traditionally has limited spatial resolution due to the diffraction of light. New advances in EU research have made it possible to bypass this barrier and overlay nanoscale data in two colours and 3D via super-resolution fluorescence microscopy (SRFM).
Chromatin seen at the molecular level
Chromatin is the complex of DNA and organizational proteins that are packed within the nucleus. The structure of chromatin is extremely important as remodelling can result in gene activation or repression. Gene control is crucial to stem cell development where a pluripotent cell can become any of a number of cell types in the body; it is vital for researchers to have precise control of these cells for stem cell therapy. Equally important is epigenetics where gene transcription can be changed by chromatin environment.

Recent research has shown that spatial organisation of chromatin is a key factor regulating gene silencing and expression. However, chromatin structure remains poorly resolved due to the nanometre length scales involved and limitations of spatial resolution, poor signal-to-noise ratio and ensemble averaging in existing methods.

Out with conventional fluorescence microscopy, in with SRFM

The lead researcher of the VCSD (Visualising chromatin structure and dynamics) project, Dr Jason Otterstrom, used Super-Resolution Fluorescence Microscopy (SRFM) to overcome these limitations with funding from a Marie Curie fellowship. With extensive experience in fluorescence microscopy applied to biological systems, he worked in two labs at the Institute for Photonic Sciences, Barcelona, first with Melike Lakadamyali and latterly with Dr Loza-Alvarez, both experts in SRFM. The technique utilised identifies the 3D position of single fluorescent dyes and reconstructs an image using these positions, similar to the pointillist or ‘dotted art’ painters of the 19th century.

The overarching goal of VCSD was to establish a new framework for characterising chromatin structure. “To do this, we had to develop a methodology and an algorithm to overlay super resolution microscopy data in two colours and in 3D,” explains Dr Otterstrom. Using the algorithm, the super resolution data helped visualise and quantify DNA together with histones on a global scale upon chromatin restructuring. The next step would be to target a defined gene loci within the nucleus to study chromatin organisation and restructuring on a local scale as it correlates with gene expression.

The search for the perfect dyes for multicolour images

Use of multiple dyes for multicolour imaging brought with it various challenges. “I found out that although some dyes are suitable for imaging some structures in single-molecule localisation-based SRFM, they do not work for other structures, such as the histones I intended to visualize,” outlines Dr Otterstrom.

The answer was to perform a wide search for appropriate dyes together with buffer conditions necessary. Finally, collaboration with another PhD student introduced the idea of using an orthogonal single-molecule method that had a different requirement for the dye quality. “I had to adjust my data workflow to put the two imaging strategies together, but it has been successful,” Dr Otterstrom reports.

Future applications on a personal basis and much wider

Analysis of the VCSD results continues as well as data recording. The methodology developed is anticipated to be adopted by researchers in stem cell and chromatin biology fields, thereby reinforcing Europe’s global reputation in scientific innovation.

“The Marie Curie fellowship enabled me to pursue applications of chromatin structural quantification as an independent biophysical researcher as well as find a satisfying job within the field,” sums up Dr Otterstrom. With the mounting importance for knowledge of chromatin structure at the nanoscale in stem cell applications and epigenetics generally, VCSD has amassed a firm knowledge base for a rapidly expanding area in biomed.

Keywords

VCSD, chromatin, super-resolution fluorescence microscopy (SRFM), stem cell, histone, pluripotent, epigenetic, orthogonal single-molecule
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