Retina tissues were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH7.4 containing 2% sucrose for 1 hour and post-fixed in 1% osmium tetroxide for 1 hour. The sample was rinsed in buffer and en-bloc stained in aqueous 2% uranyl acetate for 1 h followed by rinsing in distilled water, dehydrated in an ethanol series and infiltrated with Embed 812 (Electron Microscopy Sciences) resin. The samples were place in silicone molds and baked at 60 °C for 24 h. Hardened blocked were sectioned using a Leica UltraCut UC7. 60 nm sections were collected on formvar coated nickel grids and 250 nm sections on copper slot grids and stained using 2% uranyl acetate and lead citrate. 60 nm sections on grids were viewed FEI Tencai Biotwin TEM at 80Kv. Images were taken using Morada CCD and iTEM (Olympus) cellSens Dimension software. For electron tomography, 250 nm thick sections with 15 nm fiducial gold to aid alignment. Tomography was done using FEI Tecnai TF20 at 200Kv. Data was collected using SerialEM3.8 (Boulder) on a FEI Eagle 4Kx4K CCD camera using tilt angles −60° to 60° and reconstructed using Imod4.9 (University of Colorado Boulder).
Eagle 4k x 4k
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The Eagle 4K x 4K is a high-resolution imaging system designed for scientific applications. It features a 4096 x 4096 pixel sensor, providing a large field of view and high-quality image capture capabilities.
Automatically generated - may contain errors
3 protocols using eagle 4k x 4k
1
Ultrastructural Analysis of Retinal Tissue
2
Cryogenic Transmission Electron Microscopy of CNLV-2
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For Cryo-TEM analysis, the weighed amount of lyophilized CNLV-2 was dispersed in Milli-Q water. The dispersion was vortexed in a cyclomixture for 5 min and a little quantity of the dispersed CNLV-2 (4 µl) was taken on a clean grid. The sample was then immediately vitrified in liquid ethane followed by storage in liquid nitrogen condition until imaging [15] . Images of the sample were taken with the help of a electron microscope (Tecnai Polora, version 4.6, Netherlands) equipped with an FEI Eagle 4K x 4K charge-coupled device (CCD) camera. During imaging, vitreous grids were transferred into the electron microscope with the help of a cryostage. Throughout the experiment, the temperature of the samples was maintained at -170 °C to observe the NLVs in their native form without any degradation to the bilayer structure.
3
Cryogenic Transmission Electron Microscopy of Lipid Nanoparticles
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For Cryo-TEM analysis, weighed amount of lyophilized LNLs was dispersed in milli-Q water. The disper-sion was vortexed in a cyclomixture for 5 min and a minute quantity of the dispersed LNLs (4 µl) was taken on a clean grid. The sample was then immediately vitrified in liquid ethane followed by storage in liquid nitrogen condition until imaging. 24, (link)28 (link) Images of the sample was taken with the help of a electron microscope (Tecnai Polora, version 4.6, Netherlands) equipped with an FEI Eagle 4K x 4K charge-coupled device (CCD) camera. During imaging, vitreous grids were transferred into the electron microscope with the help of a cryostage. Throughout the experiment, the temperature of the samples was maintained -170 °C to observe the LNLs in their native form without any damage to the internal structure.
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