Monitoring chromatin remodelling in live immune cells using vibrational spectroscopy and microfluidics

Lead Research Organisation: University of Exeter
Department Name: Physics

Abstract

FTIR imaging and Raman microscopy are powerful tools for non-invasive label-free analysis of biological samples. The obtained chemical information is indicative of structure, function, and any changes e.g. due to disease occurrence, providing diagnostic capability to these spectroscopic methods.
A novel high-magnification approach to micro-FTIR imaging offers invaluable advances towards single cell detection at high spatial resolution (units of micrometre) with added chemical specificity and relatively fast measurement time (few minutes). This is especially suited for the study of micro-heterogeneous, highly structured systems such as cells under various conditions.
The immune system is a network of specialized cells and secreted signals organized in a complex yet balanced dynamic spatial arrangement. Only minor perturbations in the system could turn a normal immune response into destructive diseases, such as immunodeficiency, autoimmunity and cancer. In spite of the wealth of information we have about the immune system, it is still challenging to predict the response of a chemical stimulation of individual immune cells in vitro or in vivo. Spatially resolved readout of secreted molecules - particularly antibodies - is also difficult because of the large battery of antibodies produced and because any modifications to antibody proteins could affect their ability to neutralize invading pathogens.
Microfabrication enables the realization of microfluidic devices for the control and compartmentalization of single living cells. The fully controlled synthetic environment in the device allows the culture conditions to be altered and responses of these cells to be monitored at the scale of the individual cell. Importantly, the microfluidic devices will be fully compatible with automated FTIR imaging allowing for high-throughput measurements.
This project is aimed at developing the application of high-resolution FTIR imaging in combination with Raman microscopy to the study of live single immune cells under stress conditions in microfluidic devices. This offers the important advantage of performing in situ measurements in aqueous environment which would otherwise be affected by water absorption in the IR region if a conventional transmission approach is used.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509656/1 30/09/2016 29/09/2021
1697640 Studentship EP/N509656/1 01/02/2016 30/11/2020 Rikke Morrish
 
Description ABS Trust Bursary
Amount £150 (GBP)
Organisation Association of British Spectroscopists 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2017 
End 05/2017
 
Description CEMPS PGR Funding to attend training event
Amount £1,200 (GBP)
Organisation University of Exeter 
Department College of Engineering, Mathematics & Physical Sciences
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 07/2016
 
Description Trainee Grant
Amount € 650 (EUR)
Funding ID COST Action CA16124 
Organisation European Cooperation in Science and Technology (COST) 
Sector Public
Country Belgium
Start 01/2018 
End 01/2018
 
Title Visualisation of global chromatin architecture 
Description As an integral part of our adaptive immune system, B cells or B lymphocytes, diversify their antibody genes to produce high affinity antibodies to ward off microbes and toxins. It has been hypothesised that, prior to antibody maturation, B cells must pass through a change in the chromatin conformational state. This initiation state is a gateway connecting chromatin activation to nuclear auxeticity, i.e. expansion (contraction) of the nucleus upon stretching (compression), and nuclear auxeticity to transcriptional control via mechanotransduction. It is extremely important to understand this transient activation state given its importance for adequate cell development and function. However, this state has yet to be fully defined. Attempts to define it using molecular biology have yielded incomplete results in that there are few signatures exclusive to this state. Specifically, molecular and epigenetic signatures specifying the activation state are also observed in either a naïve or mature state. In this context, we suggest a novel and unique phenotype for the activated immune cells: they present chromatin decondensation driving nuclear auxeticity. Auxetic comes from the Greek language, meaning "which tends to increase", and auxeticity is emerging as a hot topic in materials science and physics. For example, nuclear auxeticity is a unique and remarkable property of a pre-committed state in embryonic stem cells (Nature Materials 2014). Indeed, the possibilities are enormous from a biological standpoint; the interaction between chromatin, the nuclear envelope and the cytoskeleton has been shown to alter transcription-associated responses, leading to new insights into mechanotransduction. To perform our research, we introduced a novel cutting-edge approach combining label-free hyperspectral imaging and microfluidics to show that chemically-induced chromatin decondensation, was associated with a decrease in DNA-to-protein content and the nuclei of activated B cells expanded in cross-section when stretched, i.e. they are auxetic. We validated our new technology on the fundamental biological process of cell cycle. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2019 
Provided To Others? No  
Impact The most immediate locus of impact for this work will be in the field of biology and physics, in which label-free hyperspectral imaging is emerging as a hot technique, but which have not seen molecular and nuclear properties related before in immune cells. Another area of impact will be biotechnology in that we developed novel biophysical tools to perform this research. Furthermore, the methods proposed herein could have profound implications on cellular development in general and potential on cancer transdifferentiation applications. Finally, this work will pioneer efforts to understand these transient physical changes in any cell type or synthetic material and thus will be of interest to the broad readership. 
 
Description Biological services: Mice for primary cell harvest 
Organisation University of Exeter
Department Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution I harvest primary B cells from the mice.
Collaborator Contribution The Biological Services maintain the mouse facilities and provide euthanised mice.
Impact Additional data from primary B cells to support the work undertaken with the murine B cell line CH12f3-2A.
Start Year 2017
 
Description cell_def: Python image processing project for analysing B-cell deformations in a microfluidic setup 
Organisation University of Exeter
Country United Kingdom 
Sector Academic/University 
PI Contribution I provided the experimental data for the image processing and discussed aims with Dr Jeremy Metz. After attending a Python programming workshop, I lead the optimisation of the Python image processing code with support from Jeremy.
Collaborator Contribution Dr Jeremy Metz wrote the first version of the image processing code, and further provided python programming training, and continuous support and advice.
Impact We have written modular python project for analysis of B-cell deformations in a microfluidic chip, allowing for detection and tracking of cells as they flow through a microfluidic chip. Optimisation of various parametres is ongoing. It is being tested on a range of samples. There are plans to publish the python module.
Start Year 2016
 
Description Exeter Progression in Physics: Introduction to Biophysics (Year 12 students) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact In England and Wales, only 10% of A-level physics student go on to do a degree in physics (MacDonald, A. 2004. Outreach: a guide to working with schools and colleges, the Higher Education Academy Physical Sciences Centre). Outreach is an important job for universities. It should both increase the student uptake and encourage a more diverse group of students. As part of the Widening Participation scheme, a group of Year 12 students from schools across the South West visit the university physics department to experience physics research. Two other postgraduate research students and I organised the third event; a visit to the biophysics research group. The afternoon included an introductory presentation, lab demonstrations in small groups and a 'PhD student speed dating' session. I led one of the lab demonstrations, which were all focused on real research examples. I discussed FTIR imaging with the students, using my own research to showcase the capabilities of the technique. The event feedback was positive and the students showed interest in the research. They asked questions both about the research areas/techniques, as well as requirements and expectations of a PhD student. We hope the positive feedback translates into more Physics A-level students seriously considering a career in research.
Year(s) Of Engagement Activity 2017
 
Description Invited to give the cutting edge talk at ETH/UZH in Zurich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited to give a talk at the cutting edge seminar series at the ETH/UZH on 30.10.2018
Year(s) Of Engagement Activity 2018
URL http://www.micro.biol.ethz.ch/events/immunology-seminars.html
 
Description Organising committee member for Exe-BioCon 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I was part of the organising committee for the Exe-BioCon 2016; a student-led conference for postgraduate research students. We planned the event to provide research students with an opportunity to learn skills needed for career progression. The students presented their own research to each other through posters and talks, and a panel with scientists, both from academia and industry, sparked further discussion about the importance of science communication, both to other scientists and to the general public. Based on positive feedback and interest in the event, in 2017 the conference was expanded to include postgraduate research students from the whole college of Life and Environmental Science.
Year(s) Of Engagement Activity 2016