Stimulated Raman Spectroscopy: A new tool for profiling intracellular catalysis

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Chemistry

Abstract

Understanding how fast small molecules (such as drugs) or biomolecules (such as DNA) react inside cells is crucial to the development of new medicines and the design of successful industrial processes which rely on bio-transformations. And yet at the moment there are no tools which can give both the location of a biomolecule and its rate of reaction in live cells simply relying on the inherent properties of that molecule. Current strategies either require the cell or tissue under examination to be fixed (as for histological slides) so that the concentrations of compounds can be mapped by mass spectrometry (thus not applicable for examining reactions in living cells), or rely on the incorporation of isotopes to allow detection of the molecule under study by its nuclear magnetic resonance (a technique which does not allow the position of reacting molecule within the cell to be accurately determined).
A new technique, called stimulated Raman spectroscopy (SRS), allows the vibrational properties of a molecule to be measured in a way that is:
* quantitative - allowing accurate measurement of local concentrations;
* spatially resolved - allowing molecules to be mapped inside cells;
* chemically discriminating - allowing different types of molecules to be imaged at the same time;
* and uses non-destructive optical light for detection - meaning that the studies could eventually be transferred to fibre optic technologies to be used in patients.
We propose to use SRS to study the rate of one particular reaction inside cells so that we can demonstrate that it is possible to do this both in the presence of all of the other signals from the cell, and in a way that allows us to show exactly where the reacting molecules are inside a cell.

Technical Summary

Rapid advances in the field of Raman imaging over the last decade, particularly in stimulated Raman spectroscopy (SRS), have the potential to revolutionise mechanistic understanding in biochemistry and medicinal chemistry.
* Raman is a non-destructive technique, employing relatively low energy laser irradiation, and water is only very weakly scattered in the Raman spectrum, thus live cell imaging is possible.
* Raman spectroscopy allows species-specific label-free visualisation; chemical contrast may be achieved when imaging a cell in its native environment without fixatives or stains.
* SRS can be used to give a quantitative readout (in contrast to more widely-known CARS and SERS techniques), allowing local intracellular concentrations to be determined.
In this project Raman spectroscopy will be used to measure directly the rate of reactions inside cells with spatial resolution. The thymidine analogue EdU will be incorporated into the DNA of cell nuclei and its reaction via the popular bioorthogonal copper(I) catalysed azide alkyne cycloaddition (CuAAC) click reaction will be used as the test platform. Rastering across the nucleus will allow EdU uptake to be monitored and quantified by SRS, and relative reaction rates to be mapped. The effects of enhanced local concentrations of catalyst and changes in chromatin structure on the rate of CuAAC reaction will be determined. Isotopic labelling experiments will be used to discriminate different subpopulations of the same species.

Planned Impact

We anticipate that there will be 4 major areas which will benefit from this study into the use of stimulated Raman spectroscopy for profiling the rate of intracellular reactions:

1. Academics working in the fields of bioorthogonal chemistry, biotransformations, medicinal chemistry, chromatin biology and medical imaging will benefit from the knowledge which will be gained. To ensure maximum benefit publication of the project details will be in high impact journals and knowledge gained will be communicated at national and international scientific meetings.

2. The UK capacity of personnel trained to apply an in-depth chemical knowledge of the properties of molecules to complex biological problems will be enhanced and a highly skilled worker will be produced through this programme.

3. The translational impact of this research is likely to be in the medical and industrial biotechnology industries. Industrial medicinal chemists are likely to benefit from new methods to study the uptake and metabolism of drugs, whilst the industrial biotechnology industry might benefit from a new way to follow the rate of reactions catalysed in the presence of the biomass.

4. Through outreach activities such as the Edinburgh International Science Festival and Doors Open Days public engagement with the science behind the project will be advanced.

Publications

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Vanden-Hehir S (2019) Raman Imaging of Nanocarriers for Drug Delivery. in Nanomaterials (Basel, Switzerland)

 
Description Our original objectives were met and we are currently pursuing follow-on funding to develop this work further, both in collaboration with other academics and in collaboration with industry.
Exploitation Route Stimulated Raman scattering (SRS) microscopy is a vibrational spectroscopy technique which allows the user to track cell components on the basis of specific bond vibrations alone. The techniques which we have developed could be used by the pharmaceutical industry to provide a phenotypic readout for changes in DNA accessibility upon drug treatment, as well as giving detailed knowledge about intracellular drug metabolism on the basis of changes in the intensity of specific bond vibrations.
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Science Committee - Multidisciplinary Project Award
Amount £508,640 (GBP)
Funding ID C8618/A27574 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2019 
End 04/2022
 
Description 14th Confocal Raman Imaging Symposium, Ulm, Germany, 25th - 27th September 2017. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Conference on raman imaging based at WiTEC headquarters, Ulm, Germany in September 2017.
Year(s) Of Engagement Activity 2017
URL http://www.raman-symposium.com/
 
Description 17th Royal Society of Chemistry Chemical Biology and Bioorganic Meeting, Firbush, UK, 8th-10th September 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Conference presentation titled 'SRS microscopy: Applications in label-free imaging and bioorthogonal kinetics'.
Year(s) Of Engagement Activity 2017
 
Description Chemical Biology and Drug Discovery Section of 7th EuCheMS Chemistry Congress 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk title: SRS microscopy: Applications in label-free drug imaging and bioorthogonal kinetics
Year(s) Of Engagement Activity 2018
 
Description Drug Metabolism Group Summer Meeting, Imperial College London, 15th September 2017. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Drug Metabolism Summer group eeting, Imperial College. Presented a poster at this event.
Year(s) Of Engagement Activity 2017
URL http://thedrugmetabolismgroup.com/
 
Description EaStChem Conference for Early-Career Researchers 2017, St Andrews University, 7th September 2017. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Presented a 15 min talk at this event to an audience of early career researchers and postgraduate students from the combined shools of Chemistry at Edinburgh and St Andrews University.
Year(s) Of Engagement Activity 2017
URL http://chemistry.st-andrews.ac.uk/ececr2017/
 
Description RSC Bioorthogonal and Bioresponsive Symposium, Edinburgh, 7th - 8th June 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact RSC Bioorthogonal and Bioresponsive symposium, Edinburgh
Year(s) Of Engagement Activity 2017
URL http://www.rsc.org/events/detail/24537/Bioorthogonal%20and%20Bioresponsive%2C%20a%20RSC%20symposium
 
Description Recent Advances in Synthesis and Chemical Biology XVI, University College Dublin, Ireland, 8th December 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Conference presentation titled: 'Alkynes: A Chemical Swiss Army Knife' included dissemination of results from the current research.
Year(s) Of Engagement Activity 2017
 
Description Royal Society of Chemistry Heterocyclic and Synthesis Group Postgraduate Symposium, London, UK, 20th September 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Plenary lecture at postgraduate meeting, titled: 'Alkynes: A Chemical Swiss Army Knife' included dissemination of results from the current research.
Year(s) Of Engagement Activity 2017
 
Description Speaker at the 44th Scottish Microscopy Group Meeting, Heriot-Watt University Edinburgh, 24th November 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Presented research and data from PhD studies to audience of academics, industrialists and postgraduate students. Sparked interesting questions and debate on Raman imaging techniques for intracellular drug detection.
Year(s) Of Engagement Activity 2016
URL http://scottishmicroscopygroup.org.uk/symposia/2016-symposium