Novel label-free detection technologies for 3D microscopy of biological specimens
Lead Research Organisation:
University of Southampton
Department Name: Sch of Chemistry
Abstract
There is a need for rapid microscopic imaging techniques in biomedicine. In conventional laser scanning microscopy (LSM) an excitation laser is scanned over the sample and a pixel by pixel response is detected. This is not ideal for monitoring dynamic processes. 3D imaging is also slow since such pixel by pixel scanning needs to be carried out at different stack depths. Light sheet microscopy uses planar illumination to visualise the full field of view of a sample from an orthogonal direction without raster scanning of the laser on the sample.
Light sheet microscopy is increasingly gaining popularity in biological research with all major microscope manufacturers (Leica, Zeiss, Nikon) offering light sheet microscopes. However, all current light sheet microscopes (as well as other laser scanning microscopes) are designed for detecting fluorescence. This is because typically molecules or cell structures are labelled with fluorescent endogenous proteins or exogenously added dyes/stains. Both these processes are invasive, can affect the native biochemistry and/or are only applicable to fixed (dead) specimens. Furthermore, fluorescence detection reports only those molecules which are labelled and therefore such studies do not give holistic information.
In this project the aim is to develop novel label-free detection methodologies based on scattering for light sheet microscopy. Raman and Rayleigh scattering are techniques which provide label-free information. Raman scattering, is a well-established analytical technique in chemical sciences that provides vibrational 'fingerprint' molecular information by probing the polarizability of chemical bonds. While Rayleigh scattering is also generated by interacting with molecular polarizability, the signal is emitted at the same wavelength as the incident excitation, and therefore the chemical information gets encoded only in its change of the polarisation. Thus Rayleigh polarisation anisostropy (ratio of parallel and orthogonally polarised emitted signals and also of different types of polarization as for instance circular versus linear) becomes a novel label-free measure, especially suited for imaging nearly transparent biological specimens such as cells. Size based scattering possible from some biological structures can be overcome by relying on the broadening characteristics of Rayleigh scattered light. Raman and Rayleigh light sheet imaging will thus be developed for imaging 3D biological specimens. Overall this project aims to deliver truly novel microscopic modalities implemented on a light sheet microscope. The provision of the 'highly informative' Raman and the 'simple' Rayleigh techniques on a light sheet system will be a world leading development with huge applications in biology and medicine.
Light sheet microscopy is increasingly gaining popularity in biological research with all major microscope manufacturers (Leica, Zeiss, Nikon) offering light sheet microscopes. However, all current light sheet microscopes (as well as other laser scanning microscopes) are designed for detecting fluorescence. This is because typically molecules or cell structures are labelled with fluorescent endogenous proteins or exogenously added dyes/stains. Both these processes are invasive, can affect the native biochemistry and/or are only applicable to fixed (dead) specimens. Furthermore, fluorescence detection reports only those molecules which are labelled and therefore such studies do not give holistic information.
In this project the aim is to develop novel label-free detection methodologies based on scattering for light sheet microscopy. Raman and Rayleigh scattering are techniques which provide label-free information. Raman scattering, is a well-established analytical technique in chemical sciences that provides vibrational 'fingerprint' molecular information by probing the polarizability of chemical bonds. While Rayleigh scattering is also generated by interacting with molecular polarizability, the signal is emitted at the same wavelength as the incident excitation, and therefore the chemical information gets encoded only in its change of the polarisation. Thus Rayleigh polarisation anisostropy (ratio of parallel and orthogonally polarised emitted signals and also of different types of polarization as for instance circular versus linear) becomes a novel label-free measure, especially suited for imaging nearly transparent biological specimens such as cells. Size based scattering possible from some biological structures can be overcome by relying on the broadening characteristics of Rayleigh scattered light. Raman and Rayleigh light sheet imaging will thus be developed for imaging 3D biological specimens. Overall this project aims to deliver truly novel microscopic modalities implemented on a light sheet microscope. The provision of the 'highly informative' Raman and the 'simple' Rayleigh techniques on a light sheet system will be a world leading development with huge applications in biology and medicine.
People |
ORCID iD |
| Sumeet Mahajan (Primary Supervisor) | |
| Niall Hanrahan (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| EP/R513325/1 | 01/10/2018 | 30/09/2023 | |||
| 1800765 | Studentship | EP/R513325/1 | 01/10/2016 | 31/01/2021 | Niall Hanrahan |
| Description | The project has currently resulted in a new methodology for label-free cellular imaging which combines low phototoxicity, the ability to scan quickly in 3D, and to observe changes inside cells without the use of external labels/markers. This has the potential for high impact in healthcare technology, for live imaging of certain sensitive human samples. Since the last submission, we have made a submission for protection of IP (patent) with assistance from our industrial collaborator. One journal article has been written (1st full draft), and is under in-house review before submission to a journal (coming week). Expecting publication within 6 months. A second journal article is in the final stages of the first full draft, and almost ready for internal review. |
| Exploitation Route | Further research is intended to proceed within the research group, applying the techniques in a wider variety of biological contexts to exploit the advantages it has compared to conventional imaging methods. At a later stage, others may find this label-free imaging approach useful in their own work, if a method is required that provides fast 3D imaging with a low light dose, and that is sufficiently sensitive that small changes inside a cell can be observed over time. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology,Other |
| Description | Findings from the ongoing use and refinement of the imaging methods used have been relayed to our industry collaborator, which may be used immediately to improve their product offering to both existing and new customers. As mentioned, we have now submitted a patent application to solicitors, and are waiting for a patent No. to be issued. |
| First Year Of Impact | 2018 |
| Sector | Pharmaceuticals and Medical Biotechnology,Other |
| Impact Types | Economic |
| Title | METHOD AND APPARATUS FOR OBTAINING CHEMICAL AND/OR MATERIAL SPECIFIC INFORMATION OF A SAMPLE USING LIGHT SCATTERED BY RAYLEIGH SCATTERING AND/OR RAMAN SCATTERING |
| Description | A method for obtaining chemical and/or material specific information of a sample based on scattered light. The method comprises receiving detection data comprising at least two images. Each image is indicative of the intensity of scattered light i) for incident light of a different wavelength, or ii) for incident light of a different polarization state, or iii) of a different polarization state. The scattered light comprises an elastic scattering component that is due to Rayleigh scattering of the incident light in at least a portion of the sample. Alternatively, each image is indicative of the intensity of scattered light i) of a different wavelength, or ii) for incident light of a different polarization state, or iii) of a different polarization state, wherein the scattered light comprises an inelastic scattering component that is due to Raman scattering of the incident light in at least a portion of the sample. The method further comprises determining the chemical and/or material specific information of the sample based on the change in intensity of the elastic scattering component in dependence on the change in wavelength and/or the change in polarization state of the incident and/or scattered light. |
| IP Reference | WO2021165661 |
| Protection | Patent application published |
| Year Protection Granted | 2021 |
| Licensed | No |
| Impact | IP licensing is currently under discussion with external party/parties |