For TEM imaging, monomeric and oligomeric sE2-cVLP vaccines samples were diluted to 0.2 mg/mL in PBS and adsorbed to 200-mesh-carbon-coated grids, which were stained with 2% phosphotungstic acid (pH = 7.0) for 1 min. The negatively stained sample was finally analyzed in a CM100 BioTWIN electron microscope (Phillips) at an accelerating voltage of 80 kV. Pictures were taken using an Olympus Veleta camera.
Cm 100 biotwin electron microscope
Manufactured by Philips
Sourced in Netherlands
The Philips CM 100 BioTWIN electron microscope is a high-performance laboratory instrument designed for advanced imaging and analysis. It provides users with a versatile and reliable platform for a wide range of applications in the fields of material science, life sciences, and nanotechnology. The core function of the CM 100 BioTWIN is to produce high-resolution images and data through the use of an electron beam, enabling detailed examination and characterization of samples at the nanoscale level.
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10 protocols using cm 100 biotwin electron microscope
1
Negative Staining for TEM Imaging of Vaccines
2
Negative Staining of Virus-Like Particles
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An aliquot of diluted VLPs was adsorbed to 200-mesh mica carbon-coated grids and negatively stained with 2% phosphotungstic acid (pH = 7.0). The sample was examined with a CM 100 BioTWIN electron microscope (Phillips, Amsterdam) at an accelerating voltage of 80 kV. Photographic records were performed on an Olympus Veleta camera. Particle sizes were estimated using ImageJ.
3
Negative Staining of Biological Samples
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Samples were diluted to between 0.1 and 0.3 mg/mL and adsorbed onto fresh glow-discharged 200-mesh carbon-coated grids. Grids were washed twice with ultra-pure water and stained with 2% uranyl acetate (pH 7.0) for 1 min. Excess stain was removed by blotting with filter paper and the grids were then imaged using a CM 100 BioTWIN electron microscope (Phillips, Amsterdam, The Netherlands).
4
Negative Staining for Virus-Like Particles
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Example 4
Electron Microscopy—Negative Staining
An aliquot of diluted VLPs was adsorbed to 200-mesh mica carbon-coated grids and negatively stained with 2% phosphotungstic acid (pH=7.0). The sample was examined with a CM 100 BioTWIN electron microscope (Phillips, Amsterdam) at an accelerating voltage of 80 kV. Photographic records were performed on an Olympus Veleta camera. Particle sizes were estimated using ImageJ. Representative pictures are shown in
5
Characterization of cVLP:IL-1β Vaccines
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Purified cVLP:IL-1β vaccines were quality checked by Dynamic Light Scattering (DLS) and negative stain transmission electron microscopy (TEM). For DLS analysis, vaccines were spun at 16,000× g for 2 min (Eppendorf tabletop centrifuge 5424 R) before being loaded into a disposable cuvette. The samples were run with 20 acquisitions for 7 s at 25 °C using a DynaPro Nanostar (Wyatt Technologies, Santa Barbara, CA, USA). The estimated diameter of the cVLP:IL-1β particle population and the percent polydispersity (%Pd) were calculated using Wyatt DYNAMICS software (7.10.0.21) (Wyatt Technologies). For TEM, vaccines were adsorbed onto 200-mesh carbon-coated grids and stained with 2% uranyl acetate for 1 min. The excess stain was removed with filter paper. The grids were analysed using a CM 100 BioTWIN electron microscope (Philips, Amsterdam, Netherlands).
6
Characterization of L2-Virus-Like Particles
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The 5x, 2x and 1x-L2-VLPs were assessed using negative stain transmission electron microscopy (TEM) as well as dynamic light scattering (DLS) as previously described27 (link),28 (link). Briefly, for TEM, the 5x, 2x and 1xL2-VLPs were adsorbed to 200-mesh carbon-coated grids. Grids were stained with 2% uranyl acetate (pH7.0) and analyzed with an accelerating voltage of 80 kV, using a CM 100 BioTWIN electron microscope (Phillips, Amsterdam).
For DLS measurements, the distribution of particle sizes was acquired (658 nm, at 25 °C, WYATT Technology, DynaPro NanoStar). The 5x and 2xL2-VLPs were analyzed with 20 runs and the 1xL2-VLP with 10 runs. The estimated diameter of the main particle population and the percent polydispersity (%Pd), was calculated for all three L2-VLPs.
For DLS measurements, the distribution of particle sizes was acquired (658 nm, at 25 °C, WYATT Technology, DynaPro NanoStar). The 5x and 2xL2-VLPs were analyzed with 20 runs and the 1xL2-VLP with 10 runs. The estimated diameter of the main particle population and the percent polydispersity (%Pd), was calculated for all three L2-VLPs.
7
Characterization of PCSK9 Vaccine Formulations
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Purified PCSK9 vaccines were quality assessed by DLS and negative-stain TEM. For DLS analysis, vaccines were diluted to ~0.5 mg/mL and spun at 16,000 g for 2 min before being loaded into a disposable cuvette. The samples were run with 20 acquisitions of 7 s, at 25 °C using a DynaPro Nanostar (Wyatt Technologies, Santa Barbara, CA, USA). The estimated diameter of the cVLP-PCSK9 vaccine population and percent polydispersity (%Pd) were calculated by Wyatt DYNAMICS software (v7.10.0.21, US) (Wyatt Technologies). For TEM, vaccines were adsorbed onto 200-mesh carbon-coated grids and stained with 2% uranyl acetate. The grids were analyzed using a CM 100 BioTWIN electron microscope (Philips, Amsterdam, Netherlands), with an accelerating voltage of 80 kV.
8
Negative Staining of Viral Particles
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Using the droplet method, an aliquot of VLPs was diluted to 0.2 mg/mL in PBS. Diluted VLPs were adsorbed to carbon and negatively stained with 2 % phosphotungstic acid (pH = 7.0) for 1 min. A grid was placed on the carbon floating on top of the 2 % phosphotungstic acid stain droplet. Excess stain was removed with filter paper. The sample was examined with a CM 100 BioTWIN electron microscope (Phillips) at an accelerating voltage of 80 kV. Photographic records were obtained on an Olympus Veleta camera.
9
Visualizing CSP Spy-VLP Vaccine Structure
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An aliquot of the CSP Spy-VLP vaccine was adsorbed to 200-mesh mica carbon-coated grids and negatively stained with 2% phosphotungstic acid (pH = 7.0). The VLPs were analyzed with an accelerating voltage of 80 kV, using a CM 100 BioTWIN electron microscope (Phillips, Amsterdam). An Olympus Veleta camera was utilized to obtain photographic records and particle sizes were estimated using ImageJ.
10
Characterization of Nanostructured Membranes
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The Fourier-transform infrared spectroscopy (FTIR) technique was used to investigate the chemical structure of nanostructures and prepared membranes. The scanned range was between 400 and 4000 cm−1 (WQF-510A spectrophotometer). The Field emission scanning electron microscopy (FESEM, HITACHI S-4160) and transmission electron microscopy (TEM, Philips CM 100 Biotwin Electron Microscope operated at 75 kV) analyzes were used to investigate the shape and size of the particles as well as the morphology of the membranes. To examine the surface roughness, images were prepared using an atomic force microscopy (AFM, DME-SPM Semilab). Energy dispersive X-ray (EDAX) and image mapping techniques were used to demonstrate the modification performed on KCC-1 and the dispersion of particles in the membrane matrix. The mechanical strength of the membranes was studied using a tensile test. The experiment was performed according to the ASTM D412 standard method and using a FRANK-PTI Horizontal Tensile Tester. Zeta potential test was used to measure the surface charge of membranes (HORIBA Scientific, SZ-100).
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