To generate metaphase spreads, cells were treated with hypotonic solution (0.5% sodium citrate, 0.25% potassium chloride in deionized water) and fixed in 3:1 methanol:acetic acid added drop-wise while vortexing the sample. The samples were incubated for 30 min at room temperature before centrifugation, resuspended in 10 mL of fix, and stored overnight at −20°C. The cell suspension was dropped onto high-precision coverglass (Marienfelt), stained with 50 ng/mL DAPI in PBS, and mounted in Prolong Gold (Life Technologies). Three-dimensional structured illumination microscopy (SIM) images were acquired on a N-SIM (Nikon Instruments) using a 100× 1.49 NA lens and refractive index-matched immersion oil (Nikon Instruments). SIM images were reconstructed using NiS Elements software (Nikon Instruments) from a z-stack comprised of no less than 1 µm of optical sections.
Refractive index matched immersion oil
Manufactured by Nikon
Sourced in United Kingdom
Refractive index-matched immersion oil is a specialized laboratory product designed to enhance optical imaging. Its core function is to match the refractive index of the sample, reducing refraction and distortion to improve image quality and resolution.
Automatically generated - may contain errors
Lab products found in correlation
N sim, by Nikon (4 mentions)
Du 897x 5254, by Oxford Instruments (4 mentions)
Nis elements software, by Nikon (3 mentions)
Plan apo tirf objective, by Oxford Instruments (3 mentions)
High precision cover glass, by Zeiss (3 mentions)
Nis elements software v4, by Nikon (2 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
N sim microscope, by Nikon (1 mentions)
Slowfade gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using refractive index matched immersion oil
1
Metaphase Spread Preparation and 3D-SIM Imaging
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-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).
3
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.
4
3D Structured Illumination Microscopy Protocol
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3D structured illumination microscopy (3D‐SIM) images were acquired on a Nikon N‐SIM microscope (Nikon Instruments, UK). Samples were prepared on high precision cover glass (Zeiss, Germany). Data were collected using a Nikon Plan Apo TIRF 100 × 1.49 NA objective and an Andor DU‐897X‐5254 camera using 488, 561 and 640 nm laser lines. Refractive index‐matched immersion oil (Nikon Instruments) was used for all experiments. Z‐stacks were collected with a step size of 120 nm as required by the manufacturer software. For each focal plane, 15 images were acquired (five phases, three angles) and captured using the NiS‐Elements software. SIM image processing, reconstruction and analysis were carried out using the N‐SIM module of the NiS‐Elements Advanced Research software. Images were checked for artefacts using the SIMcheck software (http://www.micron.ox.ac.uk/software/SIMCheck.php ). Images were reconstructed using NiS‐Elements software v4.6 (Nikon Instruments, Japan) from a z‐stack comprising of no < 1 μm of optical sections. In all SIM image reconstructions, the Wiener and Apodization filter parameters were kept constant.
5
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.
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