Total internal reflection fluorescence microscopy (TIR-FM) was performed as previously described.63 (link) Briefly, cells were mounted in PBS and imaged using a 60×, 1.49 NA APO TIRF objective (Nikon) mounted on a fully motorized Nikon Ti-Eclipse inverted microscope with Perfect Focus System and coupled to an Andor “Diskovery TIRF/ Borealis widefield illuminator” equipped with an additional 1.8× tube lens (yielding a final magnification of ×108). TIR-FM illumination was achieved using a Diskovery Platform (Andor Technology). For live cell experiments, cells were maintained at 37°C during imaging. Imaging sequences were acquired using a sCMOS camera with 6.5 μm pixel size (pco.edge).
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Live Cell Imaging with TIR-FM
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Corresponding Organization : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- TIR-FM illumination
- Cell imaging
- Cells mounted in PBS
- 60×, 1.49 NA APO TIRF objective (Nikon)
- Fully motorized Nikon Ti-Eclipse inverted microscope with Perfect Focus System
- Andor "Diskovery TIRF/ Borealis widefield illuminator" with 1.8× tube lens
- Diskovery Platform (Andor Technology) for TIR-FM illumination
- Live cell experiments maintained at 37°C during imaging
- SCMOS camera with 6.5 μm pixel size (pco.edge)
- Not explicitly mentioned
- Not explicitly mentioned
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