A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy
A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy
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DOI：
10.1046/j.1365-2818.2000.00774.x
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发表时间：
2000-12-01
期刊：
影响因子：
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通讯作者：
Bagshaw, CR
中科院分区：
文献类型：
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作者：
Conibear, PB;Bagshaw, CR
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 mum 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.
