Cytek Aurora Spectral Analyser: essential investment to expand the Babraham Institute Flow Cytometry capabilities, enabling pioneering bioscience

The field of flow cytometry is essential in many areas of life science research, such as immunology, haematology, cell biology and cancer studies. At BI, we study healthy ageing and Flow Cytometry is an essential tool to do this. The Flow Cytometry Core Facility is a busy, successful facility housing state of the art cytometers that are supported by dedicated and experienced staff. The Flow Cytometry Core prides itself on providing excellent service to BI scientists (over 100 use the facility) and 17 bio-tech companies who are based at Babraham Research Campus.

It is important that the equipment within the Flow Cytometry Facility is state-of-the-art to facilitate the pioneering science carried out by BI Scientists. There have been huge advances in the technology behind the flow cytometry instruments and the fluorochromes that are measured on the instruments and therefore it is important that BI Flow Cytometry Facility keeps up to date with these technology changes. One of the biggest recent changes in technology has been the introduction of spectral analysers. This equipment grant is for a Cytek Bioscience 5-laser Aurora Spectral Analyser to be housed within the BI Flow Cytometry Facility.

The Cytek Bioscience Aurora Spectral Analyser (hereafter 'Aurora'), a new spectral analyser which looks at the spectra of fluorochromes across each of its 5 lasers. This technology has been used in the field of microscopy for years but is new to flow cytometry. The analysers currently within the Core can theoretically measure 27 fluorescent markers, however it is difficult to combine more than 20 markers together due to the lack of unique commercially available dyes and the challenges of using a number of markers in combination. The Aurora allows up to 50 fluorescent parameters to be used including combinations of dyes that are impossible in conventional flow cytometry. This means that fluorochromes with similar excitation and emission maximums can be used together in experiments. This ultimately enables scientists to look at more markers from a sample, allowing them to study individual cells in more detail, reducing the size of samples needed for analysis.

Another challenge when studying cells using flow cytometry can be strong cellular autofluorescence which means it can be difficult to measure and quantify dim fluorescent markers. The Aurora measures autofluorescence as a separate parameter. Autofluorescence can then be subtracted from the fluorescence allowing scientists to detect and quantify dim fluorescent signals.

Increasing the multicolour capabilities of the BI Flow Cytometry Core Facility is essential to continue the BI world-class science. The addition of an Aurora will strengthen the capabilities of the Flow Cytometry Core Facility, which will in turn help BI scientists with their bioscience research into lifelong health and wellbeing, particularly in the areas of ageing cell immunology and regenerative medicine. Cutting-edge flow cytometry capabilities also boost our connections with Babraham Research Campus and other scientists and maximises the value of the invested equipment funding.

This application aims to enhance the multi-colour capabilities of the BI Flow Ctometry Core Facility. Flow Cytometry is an important technology for bioscience research due to its unique ability to measure individual cells looking at multiple parameters at rates of several thousand per second. This powerful technology allows scientists to carry out in-depth analysis of their cellular populations, such as immune cell activation, measuring transfection/gene editing using fluorescent protein and studying regenerative medicine. Recent advances in Cytometry technology has led to increased numbers of fluorochromes on the market, increasing the number of markers that can be analysed on each cell. However, an increase in fluorescent markers in flow cytometry can lead to problems with data spread after spectral compensation which in turn can lead to reduction in resolution of populations.

With spectral analysis, the emission spectra of each fluorescent dye is measured across all lasers, which means that dyes that are incompatible on conventional flow can be used together. The Cytek Bioscience Aurora is a 5-laser instrument which will enable BI scientists to use up to 50 fluorochromes within their experiments. This will allow more information to be gathered on each cell than is currently possible.

Many cell types have intrinsic autofluorescence which can be problematic within flow cytometry, due to a lack of resolution between autofluorescent and dimly positive cells. The Aurora measures auto-fluorescence which is then subtracted from the fluorescent signal allowing measurement and quantification of weak dyes. This is important as autofluorescence is a problem encountered by a majority of flow cytometry users at BI.

The advanced capabilities of the Aurora Spectral Analyser will allow BI Scientists to advance their bioscience research and increase their understanding of their cellular models.

The addition of a Spectral Analyser into the BI Flow Cytometry Core is expected to have a huge impact on the number of types of experiments that are carried out by not only BI Scientists but also scientists from Babraham Research Campus and external companies and academics. This will create a number of social and economic impacts. The beneficiaries of these impacts are described below. Information about the activities we will undertake to deliver these impacts are described in the 'Pathways to Impact'.

a) Contribution to scientific advancement: Additional novel technology within BI Flow Cytometry Core Facility will lead to significant scientific advancements. The Academic Beneficiaries section elaborates how research conducted within the Core will achieve this.

b) Enhancing research capacity, knowledge and skills of businesses and organisations: Unallocated capacity on all cytometers (analysers and sorters) is available for use by Campus and external companies. Any unallocated capacity on the Aurora will also be available for company use. As with BI users, company scientists will be given full training on the use of the Aurora as well as how to design panels and data analysis. As demonstrated by the letters of support from two campus companies and the two external companies mentioned in the Case for Support, there is a lot of interest from companies to use spectral analysis to enhance their research. This includes basic research as well as research into novel fluorochromes. Novel fluorochromes are essential to advance flow cytometry, therefore it is important for them to test not only on conventional flow cytometers but also spectral analysis. With the construction of two new company buildings on Campus, the use of the flow facility by companies will increase and a core equipped with state-of-the-art instruments will benefit these stakeholders.

c) Contributing to wealth creation and economic prosperity through exploitation of scientific knowledge: Through the activities of its wholly owned trading arm, Babraham Institute Enterprise, BI scientists collaborate with other organisations to translate their research. BI has a proven record of revenue generation through the exploitation of BI's IP. The use of BI's core facilities by external users increases the commercial potential and competitiveness of these companies' research programmes.

d) Enhancing efficiency, performance and sustainability of organisations: The use of unallocated capacity on our cytometers by external users, aligns with BBSRC's ethos and are supported by BBSRC Executive and Council, providing added value to their investment in BI. Bringing together BI and external researchers generates a critical mass of knowledge exchange and innovation sharing opportunities.

e) Training of skilled people: BI Flow Cytometry Core is committed and dedicated to training of staff, users and other scientists. Staff within the core are both technical specialists as well as research participants, which increases not only their knowledge but career prospects. As the Aurora is a new technology, this will give the Core Staff experience of training users on the instrument, advising on panel design and data analysis. This gives them an invaluable experience and also a unique skills set. The BI Head of Flow Cytometry, Dr Rachael Walker, was awarded a BBSRC Modular Training Partnership grant to develop training courses for industry and academic based researchers.

f) Enhancing quality of life, health and wellbeing: At BI, our research into lifelong health and well-being. Our discoveries and subsequent exploitation of IP, will contribute to improving health across the lifecourse particularly in the key areas of ageing research and regenerative medicine.