3D-SIM was performed on a DeltaVision OMX V4 Blaze fluorescence microscope (GE Healthcare) using a 60x/1.42 Oil PlanApo N UIS2 objective lens (Olympus), giving 80 nm pixel spacing in raw images with striped illumination at 3 angles and 5 phases. High precision coverslips of 170 μm thickness, and immersion oil with a refractive index of either 1.514 or 1.516 at 593 nm were used to eliminate sample induced spherical aberration, matching the measured PSF of the calibrated system, for high quality image reconstructions at around 120 nm lateral (x,y) spatial resolution with 40 nm reconstructed image pixel spacing. Fast piezo stage Z-series images were taken at 1.5-3.0 μm total thickness with 0.125 μm Z-step spacing with raw frame exposure times of 100 ms, avoiding detector saturation of the 15 bit 1.6 electron read noise pco.Edge sCMOS camera (PCO), using the mCherry/Alexa Fluor 658 fluorescence emission filter (581-636 nm) and fluorescence excitation with a 568 nm laser. Cells expressing mCherry-mamY from the mamY locus were spotted onto a 1% Mgryph agarose pad as per Toro-Nahuelpan et al (2016). 3D-SIM image reconstruction was performed in SoftWoRx (version 6.1.1, GE Healthcare) according to the method of Gustafsson and colleagues45 (link).
Oil planapo n uis2 objective
Manufactured by Olympus
The Oil PlanApo N UIS2 objective is a high-performance objective lens designed for use in microscopy applications. It features a numerical aperture of 1.4 and is optimized for use with oil immersion. The objective provides high-resolution, high-contrast imaging capabilities.
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2 protocols using oil planapo n uis2 objective
1
3D-SIM Imaging of mCherry-MamY in Cells
2
3D-SIM Imaging of mCherry-MamY in Cells
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3D-SIM was performed on a DeltaVision OMX V4 Blaze fluorescence microscope (GE Healthcare) using a 60x/1.42 Oil PlanApo N UIS2 objective lens (Olympus), giving 80 nm pixel spacing in raw images with striped illumination at 3 angles and 5 phases. High precision coverslips of 170 μm thickness, and immersion oil with a refractive index of either 1.514 or 1.516 at 593 nm were used to eliminate sample induced spherical aberration, matching the measured PSF of the calibrated system, for high quality image reconstructions at around 120 nm lateral (x,y) spatial resolution with 40 nm reconstructed image pixel spacing. Fast piezo stage Z-series images were taken at 1.5-3.0 μm total thickness with 0.125 μm Z-step spacing with raw frame exposure times of 100 ms, avoiding detector saturation of the 15 bit 1.6 electron read noise pco.Edge sCMOS camera (PCO), using the mCherry/Alexa Fluor 658 fluorescence emission filter (581-636 nm) and fluorescence excitation with a 568 nm laser. Cells expressing mCherry-mamY from the mamY locus were spotted onto a 1% Mgryph agarose pad as per Toro-Nahuelpan et al (2016). 3D-SIM image reconstruction was performed in SoftWoRx (version 6.1.1, GE Healthcare) according to the method of Gustafsson and colleagues45 (link).
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