Multiphoton fluorescence lifetime imaging: Enlightening cellular and deep tissue dynamics and mechanics.

Lead Research Organisation: University of Warwick
Department Name: Warwick Medical School


Microscopy gives us an opportunity to observe, record and study a dimension beyond the limits of our eyesight. The latest advances in microscopy means that you can watch life occurring within bacteria, plants and entire organisms such as mice the way one would watch a movie. Often this is done in biology using the principle of fluorescence. Fluorescence occurs when light, in the form of photons, are absorbed by natural occurring or synthetic chemical compounds in the and re-emitted with light of a longer wavelength. The most common usage of fluorescence in imaging biological samples occurs when the absorbed radiation is in the ultraviolet region of the spectrum, and thus invisible to the human eye, while the emitted light is in the visible region, which gives the fluorescent substance a distinct colour and can be captured using a camera. So, to image biological processes live, we often attach fluorescent molecules attached to proteins or bits of DNA that are of interest which is then excited by a high energy laser. In live microscopy, as with our hand-held cameras and phone cameras, the important part is to get the best quality images with the maximum speed. This also often means that we want to have the ability to remove out-of-focus areas to get the best picture and this remains true for imaging biological samples. Limitations exist with many fluorescence microscopy techniques when imaging thick biological samples,because it is hard to focus a laser beam on a single point deep in a tissue and collect all the emitted light thus providing a good signal-to-noise ratio. New microscopy techniques are now available which can both image very deep into the tissue and restrict the imaging to a single point of focus, which means we can get sub-cellular resolution without having stray light making the image blurry. Two-photon microscopy is one such technique which can allow imaging in very dense and thick samples where the images are generated by using two photons to excite fluorescent molecules, rather than a single photon resulting in high signal-to-noise ratio. We propose to use this technique to study process such as cell movement, cell-cell communication and sub cellular processes such as transport of cargo within the cell and physical responses of cells and tissues such as changes to their stiffness and tension. Thus, this technology will support exciting research which fits into several strategic priority areas for BBSRC and contribute to existing BBSRC projects, and planned BBSRC submissions.

Technical Summary

Imaging biological samples live over time is the cornerstone of a significant element of biomedical research. Multiphoton or Two-photon excitation is particularly useful to image biological processes in living systems at length scales ranging from sub-cellular to whole organisms. Two-photon excitation (2PE) occurs from the simultaneous absorption of two photons in a single event and is achieved by restricting excitation to a small focal volume resulting in higher signal yield for each excitation event and reduced photodamage from areas above and below the plane of focus. Furthermore, it is the gold standard to deliver Fluorescence Life Time Imaging Microscopy (FLIM), where imaging is based on the differences in the excited state decay rate from a fluorescent sample. Hence a multiphoton FLIM microscope will enable us to perform quantitative biophysical and biochemical measurements and deep tissue imaging in live cells and developing organisms over time. This will shed light on our research allowing us to visualize and analyse processes such as cell migration, tissue morphogenesis, protein-protein interactions and biophysics of development in length scales varying from bacteria to entire organisms. These research areas fit into BBSRCs Research and innovation priorities set out in the Delivery plan including (i) Advancing the frontiers of bioscience discovery: Understanding the rules of life and (ii) Tackling Strategic Challenges: Biosciences for an integrated understanding of health and (iii) Building strong foundations: People and talent, collaborations and Infrastructure.

Planned Impact

Impact summary: Economic, Societal and Training
Advances in imaging technologies as in the proposed MPFLIM in research have far reaching economic and societal impact beyond the immediate academic benefit.

Clinicians: Improving the quality of the 3D information available from imaging biological samples will inform the knowledge base of clinical practitioners, and assist in the development of therapeutic strategies. Immediate groups will be those at local hospitals including University Hospital Coventry and Warwickshire NHS Trust (UHCW).

Industry: A number of our researchers have collaborations with industry partners (Ferring pharmaceuticals with Greaves and Cytecom with Munehiro) and we expect that data generated through this technology will attract further R& D investment into research. This will further contribute to improving our quality of life, health and well-being and in the long-term contribute to creation of economic wealth. Access to the Multiphoton will also extend to Industry and contribute to their research outputs.

Training: The availability of this technology will support the wider training of both researchers, students and the technical staff managing the equipment. The technology will contribute to teaching on our 4-year PhD programmes in Warwick, including the BBSRC Midlands Integrative Training Doctoral Programme (MIBTP), contributing to the research of students from biological, mathematical and physical sciences. It will also enable us to train students to engage and absorb knowledge from different disciplines (both wet and dry lab), which will also train them for non-academic professions including pharmaceutical industries, microscope and image analysis software development. Technical staff (microscopy and data analysts) supporting the equipment will have the opportunity to broaden their own training and development, increasing their future employability.

Public: Increasing public awareness and understanding of science and its socio-economic value is key for ensuring the outputs of our science has broader impact. Since 3-D images are highly attractive to the eye, interactive data generated from this resource can be used in impact activities with groups outside of the academic arena (general public, school groups) and will make the scientific outputs more easily accessible to a lay audience.


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Description We have installed a Multiphoton microscope with FLIM capabilities that are currently highly used at the Warwick Medical School
Exploitation Route This scope has provided training to undergraduates and postgraduate students as well as research fellows and have contributed to their research work.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

Description MPE_FLIM Open Day 
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 On 24th August we hosted an open day to showcase the functionality of the MPE-FLIM system to potential users. Over 25 people signed up. During the open day, the imaging specialist handled the microscopes in person while the application specialists from both Olympus and PicoQuant drove the software remotely from Germany. The open day was an opportunity to outline the benefits of multiphoton imaging and FLIM as well as demonstrating some functionality of the system. This included two-colour imaging, fast resonant scanner imaging, simultaneous laser ablation and imaging as well as fluorescence lifetime imaging of NADH in live cells. After the open day, several potential users came forward to ask for some trial imaging, one of which is now regularly using the system.
Year(s) Of Engagement Activity 2021