Frequency-Modulated Stimulated Raman Scattering Microscopy for Label-Free Functional Imaging In-Planta

The ability to create images of living cells deep within biological tissues and to identify chemical species within them has been a dream for scientists in many disciplines for many years. This dream is becoming reality - physicists are developing cutting-edge optical microscopy techniques that allow biomedical scientists to look inside living tissues and identify biomolecules based on subtle differences in the way they interact with light.
Unfortunately, the plant science community has not been able to benefit from these techniques. Plants have evolved to be highly efficient absorbers of light, and contain pigments that generate an optical signal that overwhelms the subtle interactions between the light and the biomolecules of interest. We proposed to develop a new detection scheme that will overcome the fundamental limitations of imaging in the presence of strong optical absorption and allow both academic and industrial researchers to benefit from this cutting-edge technology.

The ability to image deep within living biological samples with sub-cellular spatial resolution and with the ability to identify chemical species has been a dream for scientists in many disciplines for many years. This dream is becoming reality. Nonlinear optical microscopy has become a powerful tool for studying living tissues due to several unique advantages over traditional methods. Moreover, techniques are emerging that allow quantitative chemically specific label-free image contrast by probing the properties of molecular components within biological tissues. These techniques derive label-free, chemically specific, image contrast based on vibrational spectroscopy and are providing scientists with a potent new form of real-time bio-imaging. Unfortunately, the plant science community has not been able to benefit from these label-free techniques due to the strong optical absorption resulting from the heavily pigmented samples. We proposed to develop a new detection scheme that will overcome the fundamental limitations of imaging in the presence of strong optical absorption and allow both academic and industrial researchers to benefit from the label-free imaging in-planta.

The proposed imaging tool will be of great benefit to researchers across a broad range of plant science related fields spanning academia and industry. We will disseminate our results through a broad range of mechanisms to ensure that we maximise engagement with all potential beneficiaries.


Beneficiaries

Short- to medium- term beneficiaries: The ability to image deep within living plant tissues samples with sub-cellular spatial resolution and with the ability to identify chemical species will be a major advance for all areas of academic research into plant science. It will enhance the capability in biochemical analysis of plants and how they respond to both environmental and pathogenic stresses, improved chemical analysis for biofuel production, and allow detailed structural analysis of plant-based materials. Beyond the plant science community, it will also allow label-free in-vivo analysis of heavily pigmented tissues such as melanoma.
R&D scientists in are as aware and as interested in the development of imaging tools to aid their research as the academic sector. Imaging techniques are used in the agrichemical industry in order to try to understand the distribution of chemicals (AIs, metabolites, adjuvants) in plants and formulations, and techniques that provide microscopic-scale resolution are of particular value. Labelling compounds is known to causes chemical perturbation that alters their uptake and movement. The ability to image compounds in a label-free manner, with chemically specific against a complex background with subcellular spatial resolution would represent a major advance in research capability for this sector.
Indirectly, laser and microscope manufacturers will benefit from the new application market that this technique will open.

Longer-term beneficiaries: In the longer term, the availability of this new optical imaging capability will lead to important insights into plants' functions that could be extremely important in the development of new crop varieties that will contribute to the development of a more intensive sustainable agriculture. It will improve our ability to monitor plant pathogens in-planta, which will lead to improved crop protect and pathogen resistance strategies. It will allow the visualisation of agrichemical interaction and translocation into plants, leading to enhanced agrichemical formulations for enhanced uptake and controlled release of pesticides.


Communication and Engagement

Academic plant science research community: We will disseminate the results of our research to the academic beneficiaries via high-profile peer-review journal publications and international conferences to ensure that the potential applications of the proposed tool researches the relevant academic research groups beyond Exeter.

Industrial R&D: We will target the relevant industry groups by hosting a workshop here at Exeter to highlight the capabilities and potential industry applications of the new imaging tool. Through current and previous collaborations with industry both the PI and Co-I are well connected within this R&D sector and, with the aid of the staff of our Research and Knowledge Transfer Team, shall draw up a list of contacts within specific industries and publicise the results of this project and the workshop event.

The General Public: The University of Exeter regularly organize talks to local schools, colleges and the public to highlight the range of research activities. These schemes also encourage participation and contribution of stakeholders' views on current and future research, and facilitate environmental and research driven activities for local, regional and national schools, organizations and other interested parties. The outputs of this project will include many highly informative images and videos, which would be particularly useful to our outreach work with local A-level and AS-level students.