Total Internal Reflection Fluorescence (TIRF) Microscopy and Single Molecule Detection


We have improved the sensitivity of ATPase assays to the single molecule detection limit by using laser-induced, total internal reflectance fluorescence microscopy. This technique allows selective excitation of fluorescent nucleotides bound to immobilised myosin molecules on the microscope slide surface. The microscope set-up was assembled from both commercial and custom-built components made in the Biomedical Joint Workshops.



Single molecule detection of a fluorescent ATP analogue (Cy3-EDA-ATP) showing binding to isolated myosin S1 molecules and its turnover with an average reaction time of about 10 seconds (record of Dr Paul B. Conibear, see reference for details).


For further technical details see: Wakelin & Bagshaw (2003) and Conibear & Bagshaw (2000). We are now developing single molecule FRET (Forster resonance energy transfer) assays to probe proteins of the Spliceosome and their interaction with RNA (Cherny et al, 2009) . See Molecular Enzymology page for more photos and Schematic for optical layout


Cubic Prism 2

Prism for generating isotropic TIRF excitation

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