Total internal reflection fluorescence microscopy (TIR‐FM) was performed as previously described.63 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).
Diskovery tirf borealis widefield illuminator
Manufactured by Oxford Instruments
The Diskovery TIRF/Borealis widefield illuminator is a lab equipment product designed to provide illumination for microscopy techniques. It offers both total internal reflection fluorescence (TIRF) and widefield illumination capabilities for various imaging applications.
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3 protocols using diskovery tirf borealis widefield illuminator
1
High-Resolution TIRF Microscopy Imaging
2
Selective Imaging of Peripheral Endosomes
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To selectively image endosomal compartments close to the cell periphery, we adjusted the illumination of TIRF evanescent field to a theoretical penetration depth of ∼200 nm (thick TIRF). Briefly, fixed 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×). TIRF illumination was achieved using a Diskovery Platform (Andor Technology). During imaging, cells were maintained at 37°C. Image sequences were acquired using a scientific CMOS camera with 6.5-µm pixel size (pco.edge).
3
Live Cell Imaging with TIR-FM
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