MRC National Institute for Medical Research, London
Science for Health
Single molecule techniques
Single molecule experiments give insights into how biological molecules work and how they are structured. Several research groups at NIMR apply and develop methods to study single biomolecules. Some of these techniques provide high resolution images of the moelcules others give dynamic information about the interactions between proteins, DNA, lipid membranes and small ligand molecules.
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TIRF (right) and Optical Tweezers (left) are powerful tools that assist studies of motor proteins which are the molecular machines contained in every cell of the body.
We have developed methods to visualise and manipulate single molecules, with high time resolution, using two laser-based techniques; Total Internal Reflection Fluorescence (TIRF) microscopy and Optical Tweezers (OT).
TIRF microscopy
TIRF microscopy uses the evanescent field associated with a totally internally reflected laser beam to excite fluorophores at the surface of a microscope coverslip. Sensitive camera systems are used to detect light emitted by the fluorophores. These measurements have a resolution of around five nanometres within 50 milliseconds.
Optical Tweezers
Optical Tweezers make use of radiation pressure to pick-up and manipulate individual molecules. Using fast detectors the position of optical trapped particles are measured with nanometre precision so that forces and movements produced by single molecules can be measured. The resolution is around one nanometre per millisecond.
Atomic Force Microscopy
Atomic Force Microscopy (AFM) enables us to analyse the structure of biological molecules by scanning their surface topology using a microfabricated mechanical probe or ‘Tip’. The AFM used at NIMR (JPK nanowizard) is ideally suited to studying biological materials in aqueous solution at room temperature. The AFM tip is scanned over the sample and deflections of the tip, as it rides over molecules fixed to the surface, are measured using a laser-based position sensor. The technique is ideally suited to studies of material for which high resolution dynamic information is required. The ultimate resolution depends on the sharpness and stiffness of the silicon tip, the mechanical properties of the specimen and also upon the mechanical stability of the laboratory and microscope system. For soft biological molecules the resolution is around five nanometres.
Atomic Force Microscopy (AFM) works by scanning an ultra sharp, silicon probe over a surface that has been sparsely coated with biological molecules or biological cells. The JPK nano-wizard used at NIMR enables simultaneous imaging by optical microscopy and AFM.
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