FCS with high countrates in TIR configuration
Hassler K., Gösch M., Anhut T., Rigler R., Lasser T.
In numerous applications of FCS the system under study is bound to a surface. Related research includes among others monitoring ligand-receptor binding kinetics on cell-surfaces or model membranes, investigating conformational changes of biomolecules (e.g. enzymes). Confocal FCS, when used to study systems on surfaces, suffers from two major drawbacks. First, the axial extend of the detection volume is approximately three times larger than its lateral extend, which leads to a background signal originating from molecules distant from the surface. Second, light reflected from the surface contributes considerably to the background.
These drawbacks can be circumvented using an excitation scheme based on TIR (Total Internal Reflection) illumination. For TIR illumination a collimated laser beam impinges on the interface between a glass substrate and an aqueous solution containing the sample, with an angle of incidence larger than the critical angle. The light is back-reflected into the glass substrate and an evanescent wave which decays rapidly with the distance to the slide is created inside the sample.
In the most commonly used realizations of TIR-FCS the sample is excited through a prism and fluorescence emission is collected via an objective situated above the sample. We have realized and investigated a new TIR-FCS configuration, which collects fluorescence emission in a more efficient way leading to a considerable increase of the SNR. We showed, that the photon count rate per molecule in TIR-FCS may come close to values obtained in confocal FCS.