BD FACSDiscover S8 Sorter with CellView Image Technology: spectral and imaging cell sorter for in-depth analysis of complex biological systems
Lead Research Organisation:
University of Cambridge
Department Name: Pathology
Abstract
This application seeks to establish a BD FACSDiscover S8 Cell Sorter with BD CellView Image Technology in the flow cytometry facility at the School of the Biological Sciences (University of Cambridge). Flow cytometry and cell sorting are complementary fundamental techniques used in a wide range of disciplines including immunology, clinical diagnostics and biomedical research. Flow cytometry allows the detection of physical and chemical parameters of a population of cells and particles, while cell sorting allows these parameters to be used to separate uniform populations according to their characteristics which can then be analysed in detail or used in further experimentation.
Traditionally, multiple fluorescent-tagged antibodies or dyes have been used to identify target populations of cells. In these conventional polychromatic flow cytometers, only a fraction of the emitted light is captured and dyes with close emission of fluorescence cannot be used together without compromising sensitivity and resolution. In addition, the complex requirements of additional detectors and filters have rendered commercial polychromatic machines expensive to both purchase and maintain. Full spectrum flow cytometers (FSFCs) emerged from these limitations and capture the entire spectrum of light emitted by fluorophores, allowing closely related dyes to be used together. Panel design can subsequently be expanded in the number of parameters assessed, enabling more detailed identification of novel populations of cells and allowing new insights into cell biology. Moreover, integrating FSFCs with cell sorters allows the separation of these novel subsets of cells for further characterisation and downstream experimentation.
The BD FACSDiscover S8 Cell Sorter with BD CellView Image Technology platform combines FSFC with sort-capable image analysis to visually confirm cells of interest. This enables cells not only to be sorted based on the proteins they express but also based on where in the cell those proteins are expressed. This opens up the exciting possibility of sorting cells based on immediate responses to cell stimuli (such as a transcription factor translocating from the cytosol to the nucleus). In addition, the equipment also allows the differentiation of cells that express the same cell markers, but that are morphologically distinct (this is particularly relevant for different myeloid cell subsets and the community studying prokaryotes and unicellular eukaryotes). The equipment will therefore substantially enhance our capacity to sort novel cell subsets for further characterisation and enable the design of CRISPR screens that could not be accomplished using existing instruments.
The research enabled by this machine also requires the capability to work with both clinical samples and those involving human pathogens. To address this, we have requested a dedicated biosecure hood that will be used to house the BD FACSDiscover S8 Cell Sorter and will ensure a wide variety of users can access the machine. This contributes to the UK's capability to carry out cutting-edge studies under biological containment and means that a wide array of users working in these subject areas can make use of the facility.
Traditionally, multiple fluorescent-tagged antibodies or dyes have been used to identify target populations of cells. In these conventional polychromatic flow cytometers, only a fraction of the emitted light is captured and dyes with close emission of fluorescence cannot be used together without compromising sensitivity and resolution. In addition, the complex requirements of additional detectors and filters have rendered commercial polychromatic machines expensive to both purchase and maintain. Full spectrum flow cytometers (FSFCs) emerged from these limitations and capture the entire spectrum of light emitted by fluorophores, allowing closely related dyes to be used together. Panel design can subsequently be expanded in the number of parameters assessed, enabling more detailed identification of novel populations of cells and allowing new insights into cell biology. Moreover, integrating FSFCs with cell sorters allows the separation of these novel subsets of cells for further characterisation and downstream experimentation.
The BD FACSDiscover S8 Cell Sorter with BD CellView Image Technology platform combines FSFC with sort-capable image analysis to visually confirm cells of interest. This enables cells not only to be sorted based on the proteins they express but also based on where in the cell those proteins are expressed. This opens up the exciting possibility of sorting cells based on immediate responses to cell stimuli (such as a transcription factor translocating from the cytosol to the nucleus). In addition, the equipment also allows the differentiation of cells that express the same cell markers, but that are morphologically distinct (this is particularly relevant for different myeloid cell subsets and the community studying prokaryotes and unicellular eukaryotes). The equipment will therefore substantially enhance our capacity to sort novel cell subsets for further characterisation and enable the design of CRISPR screens that could not be accomplished using existing instruments.
The research enabled by this machine also requires the capability to work with both clinical samples and those involving human pathogens. To address this, we have requested a dedicated biosecure hood that will be used to house the BD FACSDiscover S8 Cell Sorter and will ensure a wide variety of users can access the machine. This contributes to the UK's capability to carry out cutting-edge studies under biological containment and means that a wide array of users working in these subject areas can make use of the facility.
Technical Summary
Our application seeks to purchase a BD FACSDiscover S8 Cell Sorter with BD CellView Image Technology. This emerging technology platform combines Full Spectrum Flow Cytometry (FSFC) with sort-capable image analysis. The platform will allow users to combine full spectrum analysis with spatial and morphological insights to visually confirm cells of interest. The BD CellView Image Technology allows imaging of fast flowing cells (up to 15,000 events per second) using radio frequency-tagged emission. The system uses a 488nm laser source coupled with a beam splitter that directs the signal into an array of beamlets, each representing a single horizontal pixel in the CellView image. Each beamlet focuses on a diffraction limited spot size of 1.5 um in diameter, resulting in a pixel size of 1.5 x 1.5 um. The BD CellView optical system is integrated with the sorting module to enable the combination of imaging parameters with full-spectrum fluorescent signals in the sorting decisions. This enables cells not only to be sorted based on the proteins they express but also based on where in the cell those proteins are expressed. Hence it can be seen that this cell sorter is state-of-the-art and will significantly advance the capabilities of our cell sorting facility. The acquisition of this machine will be of great value to scientists from diverse fields across the School of the Biological Sciences.
