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| The Functional Biosystems Imaging
(FBI) group is part of STFC's Lasers for
Science Facility, operating within the Central Laser Facility
at the Rutherford Appleton Laboratory in Oxfordshire, UK. The FBI group is the home of the OCTOPUS imaging cluster. This is an advanced imaging facility operating together with the Molecular Structural Dynamics group. A range of multicolour light sources give unprecedented flexibility to combine multiple beams, multiple colours, and timing capabilities. OCTOPUS offers a range of imaging techniques including multidimensional single molecule microscopy, confocal microscopy (FLIM, FRET, and multiphoton), and optical profilometry. The modular nature of OCTOPUS allows the development and exploitation of new advanced imaging techniques as they become available, to address grand challenges in the life sciences area. The
imaging
science programme is supported with access to other techniques such as
Optical Tweezers and access to the facilities of the MSD group.
Biological laboratory facilities and expertise in sample preparation
are an integral part of the FBI's activity. |
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| OCTOPUS Imaging Cluster |
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| OCTOPUS Techniques |
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| OCTOPUS Light
Sources |
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| Highlights |
| Multidimensional (simultaneous
two-colour(FRET)/two polarisations) single molecule TIRF microscope
(live cell and protein film samples). The single molecule microscopes use total internal reflection excitation to allow the tracking of fluorescent molecules on surfaces (either dye-labelled or fluorescent proteins such as GFP). We can measure Fluorescence Resonance Energy Transfer (FRET) and polarisation (simultaneously in 4 channels of a CCD) that gives information on the distance between fluorescent molecules, so is a very powerful probe of protein interaction, or protein conformation (intra-molecular FRET) if two dyes are placed on one protein. These measurements can be made either in living cells or on isolated proteins and protein complexes. The added polarisation data give us information on the orientations of the fluorophores, essential to accurately measure distances from FRET. The combination of two colours and two polarisations is unique. Operational. |
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| 3-colour single
molecule TIRF X-Y-t tracking (Multiprotein complex kinetics and
dynamics). We are also now doing multicolour single molecule tracking, to be used to monitor the formation of multimeric protein complexes on the cell surface. This will inform on the nature of the complexes, stoichiometry, kinetics/dynamics and also on weak protein-protein interactions in physiological conditions. First of these experiments is scheduled in December. Under construction - to be scheduled next year. |
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| 5-colour single
molecule TIRF X-Y-t tracking (Multiprotein complex kinetics and
dynamics). Under construction - to be scheduled next year. |
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| Combined single molecule TIRF/NIWOP
optical profilometry (X,Y,Z,t) resolution. Under construction - to be scheduled next year. |
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Multicolour confocal fluorescence microscope with Becker & Hickl Fluorescence Lifetime Imaging (FLIM) module. The confocal microscope can be used for fluorescence lifetime imaging (FLIM), which can again be used to monitor FRET in cells, or specific conditions within cells by using environmentally sensitive probes. We currently offer three colour excitation/emission; this will be expanded to four colour in the near future. Operational. |
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Multiphoton confocal fluorescence microscope for FRET/FLIM Imaging UV to near infrared and pulse widths of nanosecond, picosecond and femtosecond. There are also cw modes available for standard confocal laser scanning microscopy Operational. |
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Support facilities. The LSF offers cell culture and biochemical preparation laboratories, and use of these labs is offered to users of OCTOPUS. |
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