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From 2012.igem.org

Detcection of VLPs

The most challenging piece of science is the detection. When producing a new VLP in vitro you don't know if it forms. So we put a lot of effort in it, to detect and visualize the Virus-Like-Particles

Electron Microscopy (EM)

One of the direct methods to detect our VLPs is with Electron Microscopy or EM. We received personally a course about the EM from Jan van Lent. In this course he explained how the EM works, how you prepare the samples and how to operate the EM. Furthermore we prepared and viewed multiple samples with our newly found skill set. This all was done in the Virology department of the Wageningen UR.

Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, electrons are being used. The same as light microscopy, electron microscopy uses multiple lenses to focus the beam so that the sample is properly lighted. The only differences with lenses are that the lenses of electron microscopy are electromagnetic instead of glass. EM has also a much greater resolution than conventional light microscopy. It is theoretically possible of around 0.005 nm, but in practice it is around 1-2 nm, this is because the lenses has certain errors, the operator (us) is inexperienced and the sample must be as this as possible. All these factors reduces the resolution of the electron microscope.

Preparation of the sample is critical to have a good resolution, during the course we learned how to do it properly. The sample must be thin, between 2 - 300nm, and must be stable in the electron microscope, this can be done by drying or cryo-freezing the sample. After drying or freezing we stain the sample with a coating. The places where there is no coating (VLPs), electron wave can go through unhindered, while the places where there are coating, electron waves break apart. This result in a picture where you can see the particles.

We checked various samples in the electron microscope. The wild types of CCMV and HepB were detected. Multiple variations of CCMV were also tested with mixed results.

Dynamic Light Scattering (DLS)

Another method to detect virus-like-particles is with dynamic light scattering also known as photon correlation spectroscopy or quasi-elastic light scattering. This technique is used to measure the size of particles and is thus an indirect way to detect particles. We used this technique as a complimentary method next to the electron microscopy.

When light travels through the sample and hits a particle, light will scatter in all directions. The dynamic light scattering machine detect the scattered light under a fixed angle (picture formation). The particles in the solution will move randomly due to Brownian motion. As the particles move , the intensity will change. This is because the scattered light can undergo constructive or deconstructive interference with the scattered light of other particles. The larger the particle, the more slower it is in Brownian motion, the slower the change of intensity is. The change in intensity over time relates to the diffusion coefficient and this diffusion coefficient is related to the particle radius using the Stokes-Einstein equation (picture).

We tested multiple samples with DLS. The main issue was that the sample was not pure enough or not concentrated enough. After optimizing the production and purification protocol we succeeded to detect VLPs.