Final Report Summary - FAPRMOL (Flow-aligned polarised Raman of biomacromolecular entities)
Summary description of the project objectives
The main aim of the project was to develop the new technique of flow-aligned polarised Raman spectroscopy. The work proceeded with two parallel lines of development, which converged towards the end of the project. The first aspect, which was the main focus of the project, was to establish the technique of Raman linear difference (RLD) spectroscopy using stretched film samples. The second aspect was to design and build a Couette flow cell for polarised Raman spectroscopy in collaboration with crystal precision optics. As an auxiliary method, ultraviolet (UV) linear dichroism (LD) had been planned to be used. Significant time has been spent on the spectral interpretation and development of the required theoretical formalism. As a complement to the RLD spectroscopy, we intended to work with the BioTOOLS ChiralRAMAN instrument to assess whether it was the correct instrument for future developments and also to collect Raman optical activity (ROA) data on a range of proteins and other biomolecules. The project was intended to conclude with a range of applications and presentation of our work in various publications formats as well as on international conferences.
Description of the work performed since the beginning of the project
Training
In the beginning of the project, the fellow was trained in number of spectroscopy methods in order to gain the expertise necessary to the development of the new Raman-based spectroscopy method. Training included LD of different types of deoxyribonucleic acid (DNA) as well as fluorescence measurements of these compounds. The fellow undertook work to develop a fluorescence instrument for fluorescence detected LD since this instrument has many features in common with a Raman instrument. The professional skills of the fellow have been enriched with synchrotron radiation LD technique experience (Aarhus, Denmark). A crucial part of the training was gaining the necessary expertise in Raman techniques firstly using unpolarised Raman and then polarised Raman.
New technique development: stretched film RLD spectroscopy
The project began with examining a range of sample orientation methods used in other forms of spectroscopy and testing them with Raman spectroscopy. We decided to adapt the stretched polyethylene film method used for UV-visible LD spectroscopy. With this approach we have been able to collect artefact-free RLD spectra and Raman linear sum (RLS) spectra of a range of small molecules (nucleic acids, vitamin B2 derivatives, steroids, poly-aromatic carbohydrates). We designed and built a film stretcher specifically for this purpose.
COUETTE flow cell design for RLD
In the early stages of the project a Couette flow cell designed for available solution-phase Raman instruments was designed and commissioned. It was finally built by crystal precision optics in month 18. This cell has been tested in the final 6 months mainly on a Thermonicolet NXR 9650 Fourier-transform (FT) Raman spectrometer at Reading University. Samples included DNA and carbon nanotubes and we concluded that the cell was appropriate for measuring flow-oriented Raman spectra. Unfortunately, the stability of the Thermonicolet spectrometer was not sufficient to measure sequential parallel polarised and perpendicular polarised spectra to determine the RLD as the difference. A collaboration has therefore been established with BioTOOLS to develop a new instrument based on their ROA instrument. This instrument has been designed and will measure the two component spectra simultaneously. Funding needs to be found to enable the instrument to be built.
Data analysis and interpretation
Once the first RLD spectra were collected it became apparent that a new theoretical approach was needed to describe what was being measured. This was not in the original workplan but was undertaken as part of the project and has been completed. Collaboration with Dr J. Cheeseman of Gaussian Inc. was established and the Gaussian code rewritten to enable it to calculate RLD spectra according to the theory derived for this work.
Description of the main results achieved so far
The main results of the project are:
(1) development of the new technique of RLD spectroscopy;
(2) development of a new sample matrix (polyethylene) for Raman spectroscopy;
(3) design, building and application of a film stretcher for RLD;
(4) design and build of a Couette flow cell and its testing for Raman spectroscopy;
(5) collection and interpretation of RLD spectra for a range of small molecule samples;
(6) derivation of theory and its computational implementation in collaboration with Gaussian Inc.
Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
As a longer-term benefit, development of RLD spectroscopy in solution is planned. In the near future, an instrument suitable for that technique will be designed and built. We believe that the new technique will be particularly useful in studies of bacteria cell membranes. We expect that the commercial version of RLD instrumentation will be available soon for the wider public.
During the whole stay in Warwick University the fellow had regular meetings with her supervisor in order to monitor the progress of the project. She also participated in the number of conferences, meetings and transferable skills trainings. A number of grant applications has been written.
The main aim of the project was to develop the new technique of flow-aligned polarised Raman spectroscopy. The work proceeded with two parallel lines of development, which converged towards the end of the project. The first aspect, which was the main focus of the project, was to establish the technique of Raman linear difference (RLD) spectroscopy using stretched film samples. The second aspect was to design and build a Couette flow cell for polarised Raman spectroscopy in collaboration with crystal precision optics. As an auxiliary method, ultraviolet (UV) linear dichroism (LD) had been planned to be used. Significant time has been spent on the spectral interpretation and development of the required theoretical formalism. As a complement to the RLD spectroscopy, we intended to work with the BioTOOLS ChiralRAMAN instrument to assess whether it was the correct instrument for future developments and also to collect Raman optical activity (ROA) data on a range of proteins and other biomolecules. The project was intended to conclude with a range of applications and presentation of our work in various publications formats as well as on international conferences.
Description of the work performed since the beginning of the project
Training
In the beginning of the project, the fellow was trained in number of spectroscopy methods in order to gain the expertise necessary to the development of the new Raman-based spectroscopy method. Training included LD of different types of deoxyribonucleic acid (DNA) as well as fluorescence measurements of these compounds. The fellow undertook work to develop a fluorescence instrument for fluorescence detected LD since this instrument has many features in common with a Raman instrument. The professional skills of the fellow have been enriched with synchrotron radiation LD technique experience (Aarhus, Denmark). A crucial part of the training was gaining the necessary expertise in Raman techniques firstly using unpolarised Raman and then polarised Raman.
New technique development: stretched film RLD spectroscopy
The project began with examining a range of sample orientation methods used in other forms of spectroscopy and testing them with Raman spectroscopy. We decided to adapt the stretched polyethylene film method used for UV-visible LD spectroscopy. With this approach we have been able to collect artefact-free RLD spectra and Raman linear sum (RLS) spectra of a range of small molecules (nucleic acids, vitamin B2 derivatives, steroids, poly-aromatic carbohydrates). We designed and built a film stretcher specifically for this purpose.
COUETTE flow cell design for RLD
In the early stages of the project a Couette flow cell designed for available solution-phase Raman instruments was designed and commissioned. It was finally built by crystal precision optics in month 18. This cell has been tested in the final 6 months mainly on a Thermonicolet NXR 9650 Fourier-transform (FT) Raman spectrometer at Reading University. Samples included DNA and carbon nanotubes and we concluded that the cell was appropriate for measuring flow-oriented Raman spectra. Unfortunately, the stability of the Thermonicolet spectrometer was not sufficient to measure sequential parallel polarised and perpendicular polarised spectra to determine the RLD as the difference. A collaboration has therefore been established with BioTOOLS to develop a new instrument based on their ROA instrument. This instrument has been designed and will measure the two component spectra simultaneously. Funding needs to be found to enable the instrument to be built.
Data analysis and interpretation
Once the first RLD spectra were collected it became apparent that a new theoretical approach was needed to describe what was being measured. This was not in the original workplan but was undertaken as part of the project and has been completed. Collaboration with Dr J. Cheeseman of Gaussian Inc. was established and the Gaussian code rewritten to enable it to calculate RLD spectra according to the theory derived for this work.
Description of the main results achieved so far
The main results of the project are:
(1) development of the new technique of RLD spectroscopy;
(2) development of a new sample matrix (polyethylene) for Raman spectroscopy;
(3) design, building and application of a film stretcher for RLD;
(4) design and build of a Couette flow cell and its testing for Raman spectroscopy;
(5) collection and interpretation of RLD spectra for a range of small molecule samples;
(6) derivation of theory and its computational implementation in collaboration with Gaussian Inc.
Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
As a longer-term benefit, development of RLD spectroscopy in solution is planned. In the near future, an instrument suitable for that technique will be designed and built. We believe that the new technique will be particularly useful in studies of bacteria cell membranes. We expect that the commercial version of RLD instrumentation will be available soon for the wider public.
During the whole stay in Warwick University the fellow had regular meetings with her supervisor in order to monitor the progress of the project. She also participated in the number of conferences, meetings and transferable skills trainings. A number of grant applications has been written.