Photoswitchable dyes for super-resolution microscopy
Lead Research Organisation:
University of Oxford
Abstract
This project falls under the EPSRC synthetic supramolecular chemistry, and sensors and instrumentation research areas.
Super-resolution microscopy is an invaluable tool for biological researchers seeking to visualise and understand the complex intracellular environment, which was awarded the Nobel Prize in Chemistry in 2014. It allows the resolution of objects below the diffraction limit of light, giving unprecedented detail of the structure and function of cells and the processes behind health and disease. Super-resolution microscopy requires fluorescent dyes with unique photophysical properties that are applied to cells in order to light up certain structures or molecules.
Suitable fluorescent dyes for super-resolution imaging need to switch between a bright 'on' state, and a dark 'off' state. One way to design this function is to make molecules that consist of a fluorescent dye, and a separate, reversible quenching unit that can turn the dye on and off in response to light. There are a number of very high performing fluorescent dyes with a range of properties that can be tailored for specific applications, however the range of photoswitchable quenchers is much smaller, and therefore the limiting aspect of molecular design. This project will focus on the following systems as ways of reversibly quenching fluorescence:
1. FRET-based photoswitchable emission quenchers such as spiroxazines and diarylethenes. These molecules switch between an open and a closed form, triggered by light, which changes their absorption properties between a form that overlaps with the emission of the dye (thereby quenching it), one that does not.
2. Mechanical photoswitches such as azobenzenes, which do not directly quench emission but undergo a geometric change that modifies the distance between a fluorophore and a separate quenching unit.
This project will involve the design and synthesis of supramolecular systems in these two categories, to fulfil the criteria needed for a useful super-resolution imaging tool. It will also include undertaking computational studies in order to validate the chosen designs before synthesis. The photophysical properties of the dyes will be measured to ensure molecules have the appropriate characteristics for imaging applications. At this stage, computational studies will aid in understanding the basic structure-photophysical property relationships, and support future improvements to the design. Successful candidate molecules will then be tested using super-resolution microscopy with Christian Eggeling's group at the Weatherall Institute of Molecular Medicine to validate their performance in vitro.
Super-resolution microscopy is an invaluable tool for biological researchers seeking to visualise and understand the complex intracellular environment, which was awarded the Nobel Prize in Chemistry in 2014. It allows the resolution of objects below the diffraction limit of light, giving unprecedented detail of the structure and function of cells and the processes behind health and disease. Super-resolution microscopy requires fluorescent dyes with unique photophysical properties that are applied to cells in order to light up certain structures or molecules.
Suitable fluorescent dyes for super-resolution imaging need to switch between a bright 'on' state, and a dark 'off' state. One way to design this function is to make molecules that consist of a fluorescent dye, and a separate, reversible quenching unit that can turn the dye on and off in response to light. There are a number of very high performing fluorescent dyes with a range of properties that can be tailored for specific applications, however the range of photoswitchable quenchers is much smaller, and therefore the limiting aspect of molecular design. This project will focus on the following systems as ways of reversibly quenching fluorescence:
1. FRET-based photoswitchable emission quenchers such as spiroxazines and diarylethenes. These molecules switch between an open and a closed form, triggered by light, which changes their absorption properties between a form that overlaps with the emission of the dye (thereby quenching it), one that does not.
2. Mechanical photoswitches such as azobenzenes, which do not directly quench emission but undergo a geometric change that modifies the distance between a fluorophore and a separate quenching unit.
This project will involve the design and synthesis of supramolecular systems in these two categories, to fulfil the criteria needed for a useful super-resolution imaging tool. It will also include undertaking computational studies in order to validate the chosen designs before synthesis. The photophysical properties of the dyes will be measured to ensure molecules have the appropriate characteristics for imaging applications. At this stage, computational studies will aid in understanding the basic structure-photophysical property relationships, and support future improvements to the design. Successful candidate molecules will then be tested using super-resolution microscopy with Christian Eggeling's group at the Weatherall Institute of Molecular Medicine to validate their performance in vitro.
Planned Impact
This programme is focused on a new cohort-driven approach to the training of next-generation doctoral scientists in the practice of novel and efficient chemical synthesis coupled with an in-depth appreciation of its application to biology and medicine.
This collaborative academic-industrial SBM CDT will have long-term benefit to the chemical industry, including the pharmaceutical, agrochemical and fine chemical sectors. These industries will benefit through: (i) the potential to employ individuals trained with broad and relevant scientific and transferable skills; (ii) new approaches to the investigation of complex biological and medical problems through novel chemistry; and (iii) better and more efficient synthetic methods.
We will link the work of DSTL, and our pharmaceutical and agrochemical partners (GSK, UCB, Vertex, Evotec, Eisai, AstraZeneca, Syngenta, Novartis, Takeda, Sumitomo and Pfizer) through a common theme of synthesis training. The design and synthesis of new compounds is essential for disease treatment and prevention, and for maintaining food security. Synthesis contributes significantly to UK tax revenue and results in sustained employment across a number of sectors. Employers in the finance, law, health, academic, analytical, government, and teaching professions, for example, also recognise the value of the translational skill-sets possessed by synthesis postgraduates, which this programme will provide.
The SBM CDT training programme will adopt an IP-free model to enable completely free exchange of information, know-how and specific expertise between students and supervisors on different projects and across different industrial companies. This will lead to better knowledge creation through unfettered access to information from all academics, partners and students involved in the project. By focussing on basic science, we will engender genuine collaboration leading to enabling technology that will be of use across a wide range of industries.
We will train the next generation of multidisciplinary synthetic chemists with an appreciation of the impact of synthesis in biology and medicine. Their unconstrained view of synthesis will aid in new scientific discoveries leading to new products, which (with appropriate inward investment), can lead to the formation of new companies and new UK employment.
We will, in part through an alliance with the Defence, Science and Technology Laboratory, engage with policy-makers to influence future policy issues involving chemistry such as food security and the rise of antibiotic resistance (both of which are relevant to our programme and are important for society as a whole).
Outreach and public engagement will be a key aspect of our programme; and all students in the proposed SBM CDT will take part in at least one outreach activity. Typical activities include: open days in the Chemistry Department through the 'Outreach Alchemists', engaging with the Oxfordshire Science Festival and participation in the various other activities already in place through the public engagement programme of the Department of Chemistry.
The research output of the students will be disseminated via high impact international publications and lectures; these will be of value to other academics in relevant fields and will be of value in the development of further research funding applications. Outreach activities and research output will also be advertised on a website dedicated to the proposed SBM programme.
This collaborative academic-industrial SBM CDT will have long-term benefit to the chemical industry, including the pharmaceutical, agrochemical and fine chemical sectors. These industries will benefit through: (i) the potential to employ individuals trained with broad and relevant scientific and transferable skills; (ii) new approaches to the investigation of complex biological and medical problems through novel chemistry; and (iii) better and more efficient synthetic methods.
We will link the work of DSTL, and our pharmaceutical and agrochemical partners (GSK, UCB, Vertex, Evotec, Eisai, AstraZeneca, Syngenta, Novartis, Takeda, Sumitomo and Pfizer) through a common theme of synthesis training. The design and synthesis of new compounds is essential for disease treatment and prevention, and for maintaining food security. Synthesis contributes significantly to UK tax revenue and results in sustained employment across a number of sectors. Employers in the finance, law, health, academic, analytical, government, and teaching professions, for example, also recognise the value of the translational skill-sets possessed by synthesis postgraduates, which this programme will provide.
The SBM CDT training programme will adopt an IP-free model to enable completely free exchange of information, know-how and specific expertise between students and supervisors on different projects and across different industrial companies. This will lead to better knowledge creation through unfettered access to information from all academics, partners and students involved in the project. By focussing on basic science, we will engender genuine collaboration leading to enabling technology that will be of use across a wide range of industries.
We will train the next generation of multidisciplinary synthetic chemists with an appreciation of the impact of synthesis in biology and medicine. Their unconstrained view of synthesis will aid in new scientific discoveries leading to new products, which (with appropriate inward investment), can lead to the formation of new companies and new UK employment.
We will, in part through an alliance with the Defence, Science and Technology Laboratory, engage with policy-makers to influence future policy issues involving chemistry such as food security and the rise of antibiotic resistance (both of which are relevant to our programme and are important for society as a whole).
Outreach and public engagement will be a key aspect of our programme; and all students in the proposed SBM CDT will take part in at least one outreach activity. Typical activities include: open days in the Chemistry Department through the 'Outreach Alchemists', engaging with the Oxfordshire Science Festival and participation in the various other activities already in place through the public engagement programme of the Department of Chemistry.
The research output of the students will be disseminated via high impact international publications and lectures; these will be of value to other academics in relevant fields and will be of value in the development of further research funding applications. Outreach activities and research output will also be advertised on a website dedicated to the proposed SBM programme.
Organisations
People |
ORCID iD |
| Harry Anderson (Primary Supervisor) | |
| Kathryn Leslie (Student) |