Super-resolution images were acquired using structured illumination microscopy. Samples were prepared on high precision cover-glasses (Zeiss, Germany). 3D SIM images were acquired on an N-SIM (Nikon Instruments, UK) instrument using a 100×1.49 NA lens and refractive index-matched immersion oil (Nikon Instruments). Samples were imaged using a Nikon Plan Apo TIRF objective (NA 1.49, oil immersion) and an Andor DU-897X-5254 camera using 405, 488 and 561 nm laser lines. Step size for Z-stacks was set to 0.120 μm as required by the manufacturer's software. For each focal plane, 15 images (five phases, three angles) were captured with the NIS-Elements software. SIM image processing, reconstruction and analysis were carried out using the N-SIM module of the NIS-Element Advanced Research software. Images were checked for artifacts using the SIMcheck software (http://www.micron.ox.ac.uk/software/SIMCheck.php ). Images were reconstructed using NiS Elements software (Nikon Instruments) from a z-stack comprising at least 1 µm of optical sections. In all SIM image reconstructions the Wiener and Apodization filter parameters were kept constant. 3D datasets were visualized and analyzed using Imaris V8.4 (Bitplane, Oxford Instruments, UK).
Plan apo tirf objective
Manufactured by Oxford Instruments
The Plan Apo TIRF objective is a high-numerical aperture objective lens designed for total internal reflection fluorescence (TIRF) microscopy. It is optimized to provide a large field of view and excellent image quality while maintaining the critical angle required for TIRF illumination.
Automatically generated - may contain errors
Lab products found in correlation
Du 897x 5254, by Oxford Instruments (4 mentions)
N sim, by Nikon (3 mentions)
Refractive index matched immersion oil, by Nikon (3 mentions)
Nis elements software, by Nikon (2 mentions)
High precision cover glass, by Zeiss (2 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Nis elements software v4, by Nikon (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Quantitation module of volocity, by PerkinElmer (1 mentions)
4 protocols using plan apo tirf objective
1
Super-Resolution Imaging Using SIM
2
Super-Resolution Imaging Using SIM
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Super-resolution images were acquired using structured illumination microscopy. Samples were prepared on high precision cover-glass (Zeiss, Jena, Germany). 3D SIM images were acquired on an N-SIM (Nikon Instruments, UK) using a ×100 1.49NA lens and refractive index matched immersion oil (Nikon Instruments). Samples were imaged using a Nikon Plan Apo TIRF objective (NA 1.49, oil immersion) and an Andor DU-897X-5254 camera using a 488 nm laser line. Z-step size for z stacks was set to 0.120 µm as required by the manufacturer’s software. For each focal plane, 15 images (5 phases, 3 angles) were captured with the NIS-Elements software. SIM image processing, reconstruction and analysis were carried out using the N-SIM module of the NIS-Element Advanced Research software. Images were checked for artefacts using the SIMcheck software (www.micron.ox.ac.uk/software/SIMCheck.php ). Images were reconstructed using NiS Elements software (v4.6, Nikon Instruments) from a z stack comprising of no less than 1 µm of optical sections. In all SIM image reconstructions, the Wiener and Apodization filter parameters were kept constant.
3
Superresolution Imaging of Ovarian Samples
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Superresolution images of immunostained ovaries mounted in SlowFade Gold antifade reagent (Invitrogen S36936) were acquired using structured illumination microscopy (SIM). Samples were prepared on high precision cover glass (Zeiss). 3D SIM images were acquired on an N-SIM (Nikon Instruments) using a 100× 1.49-NA lens and refractive index–matched immersion oil (Nikon Instruments). Samples were imaged using a Nikon Plan Apo TIRF objective (NA 1.49, oil immersion) and an Andor DU-897X-5254 camera using 405-, 488-, 561-, and 640-nm laser lines. Z-step size for Z stacks was set to 0.120 µm as required by manufacturer software. For each focal plane, 15 images (five phases, three angles) were captured with the NIS-Elements software. SIM image processing, reconstruction, and analysis were performed using the N-SIM module of the NIS-Element Advanced Research software. Images were checked for artifacts using the SIM check software (http://www.micron.ox.ac.uk/software/SIMCheck.php ). Images were reconstructed using NiS Elements software v4.6 (Nikon Instruments) from a z-stack comprising ≥1 µm of optical sections. In all SIM image reconstructions, the Wiener and Apodization filter parameters were kept constant.
4
Structured Illumination Microscopy Imaging
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Images were acquired using structured illumination microscopy (SIM) performed on an Eclipse Ti inverted microscope equipped with a Nikon Plan Apo TIRF objective (NA 1.49, oil immersion) and an Andor DU-897X-5254 camera. Laser lines at 405, 488 and 561 nm were used. Step size for z-stacks was set to 0.120 μm, which is well within the Nyquist criterion. For each focal plane, 15 images (five phases, three angles) were captured with the NIS-Elements software. SIM image processing and reconstruction were carried out using the N-SIM module of the NIS-Element Advanced Research software. Image analysis was carried out using the Quantitation module of Volocity (PerkinElmer) with x and y binning resolution of 32 nm.
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