A comparison of optical geometries for combined flash photolysis and totalinternal reflection fluorescence microscopy

Total internal reflection fluorescence (TIRF) microscopy, used in conjuncti on with flash photolysis, provides a useful way of investigating the kineti cs of macromolecular interactions. We compare different TIRF optical geomet ries to establish an optimal combination. Excitation light was introduced v ia four different arrangements: (1) a prism positioned on the microscope op tical axis, (2) an offset prism with propagation through a silica slide tra ns to the objective lens, (3) an offset prism with propagation through a si lica coverslip cis to a water-immersion objective lens and (4) a prismless arrangement using a high NA oil-immersion objective lens. Photolysis was ac hieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sampl e. Although the offset trans prism gave the best signal-to-background ratio , a customised thin rhombic prism incorporated, on axis, into the flash con denser assembly was almost as good and was more practical for scanning mult iple fields. An oil-immersion lens gave the brightest image for sample dept hs < 30 mum but above this limit, a water-immersion lens was better. The pr ismless arrangement may offer advantages in other situations but it is impo rtant to check the actual numerical aperture of the objective lens.