A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy.

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water-immersion objective lens and (4) a prismless arrangement using a high NA oil-immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal-to-background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil-immersion lens gave the brightest image for sample depths < 30 micrometer but above this limit, a water-immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.