FLIM imaging for intermolecular FRET experiments (Fig. 2e –f ) was performed on a Nikon Ti Eclipse Inverted confocal microscope with a Plan Apo Lambda 60✕/1.40 Oil objective. The confocal microscope is equipped with a Picoquant Laser Scanning microscope TCSPC Upgrade with SymPhoTime 64. Samples were excited with a 40 MHz pulsed 485 ± 10 nm laser. The laser light was reflected using a 560 nm dichroic filter and the detector collected emitted photons that passed a 582/75 nm bandpass filter. Tension FLIM (Fig. 2l ) was performed on Timebow imaging on Abberior STED microscope equipped with 60✕/1.40 Oil objective, two pulsed STED lasers (595 and 775 nm), four excitation lasers (405, 485, 561, 640 nm) and a MATRIX array detector.
Plan apo lambda 60 1.40 oil objective
Manufactured by Nikon
The Plan Apo Lambda 60×/1.40 Oil objective is a high-performance microscope objective designed for a variety of microscopy applications. It features a magnification of 60x and a numerical aperture of 1.40, making it suitable for high-resolution imaging. The objective is optimized for use with immersion oil.
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3 protocols using plan apo lambda 60 1.40 oil objective
1
Fluorescence Lifetime Imaging for Intermolecular FRET
2
Fluorescence Microscopy Imaging of Cells
3
Fluorescence Microscopy Imaging of Cells
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Cells were seeded onto functionalized coverslips at a density of 2-3000 cells cm -2 and incubated at 37°C in 5% CO 2 atmosphere for 20-30 min before fluorescence microscopy imaging. Fluorescence microscopy was carried out using a Nikon Eclipse Ti driven by the NIS Elements Advanced Research software. The microscope objective used was CFI Apo 100X (numerical aperture 1.49) objective (Nikon). The optical system includes a and a total internal reflectance fluorescence (TIRF) variable mirror launcher and a Nikon Perfect Focus System, an interferometry-based device which corrects z-drift of the stage. Fluorescence excitation of samples through the optical system was carried out 488nm (50 mW) and 561nm (50 mW) lasers. Fluorescence micrographs of IFP 2.0-paxillin were acquired using a LED source passed through a Cy5 fluorescence filter cube (Chroma 49006 filter set). The camera used to collect images was a Andor DU-897 X-9319 camera imaging at 50-500 ms exposure times. FLIM images were acquired using a Nikon Ti Eclipse Inverted Laser Scanning confocal microscope with a Plan Apo Lambda 60×/1.40 Oil objective equipped with Picoquant Time Correlated Single Photon Counting (TCSPC) Upgraded with SymPhoTime 64 2.1.3813. TCSPC. Average fluorescence lifetime per pixel was determined using the SymPhoTime Fast FLIM algorithm.
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