Confocal microscopy was performed using a Nikon A1R laser-scanning confocal microscope equipped with 60×/1.4 NA and 100×/1.49 NA objectives. SIM was performed using a Nikon N-SIM with an Apo TIRF 100×/1.49 NA objective. All images used for quantitative comparisons were prepared with equal treatment, acquired with identical parameters (e.g. pinhole diameter, detector gain), and processed in an identical manner. Richardson–Lucy deconvolution of image volumes (20 iterations) was performed using Nikon Elements software. Live-cell imaging of W4 cells was performed on a Nikon Yokogawa CSU-X1 spinning-disk confocal microscope. Images were contrast-enhanced and cropped using ImageJ software (National Institutes of Health).
Tirf 100 1.49 na objective
Manufactured by Nikon
The TIRF 100×/1.49 NA objective is a high-numerical aperture objective lens designed for Total Internal Reflection Fluorescence (TIRF) microscopy. It provides a large numerical aperture of 1.49, which is suitable for exciting fluorophores in a thin layer close to the coverslip surface.
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2 protocols using tirf 100 1.49 na objective
1
Multimodal Microscopy Techniques Protocol
2
Imaging Dynein Localization in Yeast
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Mid log-phase S. cerevisiae cells were mounted on an agarose pad made from SC media. For Figures 6 and 7 , live cells endogenously modified to express fluorescently labeled DYN1-3XGFP, CFP-TUB1, and SPC110-tdTomato were imaged using a Yokogawa W1 confocal scanhead mounted to a Nikon Ti2 microscope with an Apo TIRF 100 × 1.49 NA objective. The microscope was run with NIS Elements using the 488 nm and 561 nm lines of a six-line (405 nm, 445 nm, 488 nm, 515 nm, 561 nm, and 640 nm) LUN-F-XL laser engine and Prime95B cameras (Photometrics). The localization of DYN1-3XGFP foci was assessed and the number of foci on each location (SPB, microtubule plus end, and cell cortex) was counted.
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