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A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy
Author(s) -
Conibear P. B.,
Bagshaw C. R.
Publication year - 2000
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1046/j.1365-2818.2000.00774.x
Subject(s) - total internal reflection fluorescence microscope , optics , lens (geology) , microscope , numerical aperture , materials science , total internal reflection , prism , fluorophore , microscopy , laser , oil immersion , fluorescence , physics , wavelength
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 µm 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.