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Team:Wageningen UR/MethodsDetection
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The most straight forward method to detect VLPs is Electron Microscopy (EM). We prepared and investigated multiple samples ourselves. This work was done at the Virology department of the Wageningen University. | The most straight forward method to detect VLPs is Electron Microscopy (EM). We prepared and investigated multiple samples ourselves. This work was done at the Virology department of the Wageningen University. | ||
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Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, it is done by electrons. The EM has multiple lenses to focus the beam to magnify and light the sample. The lenses are electromagnetic instead of glass and therefore allow a much higher resolution than conventional light microscopy. In theory it is possible to reach a resolution of around 0.005 nm, but in practice it is mostly around 1-2 nm. Possible reasons are certain errors of the lenses, inexperience of the operator (for example our teammembers) or the sample is not thin enough. All these factors can reduce the resolution of the electron microscope. We investigated various samples obtained in our experiments with the help of the EM. The wild types of the Cowpea chlorotic mottle virus (CCMV) and Hepatitis B (HepB) were detected. Multiple variations of CCMV have been tested too, unfortunately with mixed results. [add pictures] | Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, it is done by electrons. The EM has multiple lenses to focus the beam to magnify and light the sample. The lenses are electromagnetic instead of glass and therefore allow a much higher resolution than conventional light microscopy. In theory it is possible to reach a resolution of around 0.005 nm, but in practice it is mostly around 1-2 nm. Possible reasons are certain errors of the lenses, inexperience of the operator (for example our teammembers) or the sample is not thin enough. All these factors can reduce the resolution of the electron microscope. We investigated various samples obtained in our experiments with the help of the EM. The wild types of the Cowpea chlorotic mottle virus (CCMV) and Hepatitis B (HepB) were detected. Multiple variations of CCMV have been tested too, unfortunately with mixed results. [add pictures] | ||
Revision as of 11:20, 24 September 2012
Contents |
Detection of VLPs
A key part of our project is the detection of VLPs. We need sufficient visualization to get conclusive evidence of VLP formation. Besides, we will investigate alternative methods to detect the formation and stability of Virus-Like Particles.
Electron Microscopy (EM)
The most straight forward method to detect VLPs is Electron Microscopy (EM). We prepared and investigated multiple samples ourselves. This work was done at the Virology department of the Wageningen University.
Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, it is done by electrons. The EM has multiple lenses to focus the beam to magnify and light the sample. The lenses are electromagnetic instead of glass and therefore allow a much higher resolution than conventional light microscopy. In theory it is possible to reach a resolution of around 0.005 nm, but in practice it is mostly around 1-2 nm. Possible reasons are certain errors of the lenses, inexperience of the operator (for example our teammembers) or the sample is not thin enough. All these factors can reduce the resolution of the electron microscope. We investigated various samples obtained in our experiments with the help of the EM. The wild types of the Cowpea chlorotic mottle virus (CCMV) and Hepatitis B (HepB) were detected. Multiple variations of CCMV have been tested too, unfortunately with mixed results. [add pictures]
EM Course
The goal of the course was to get familiar with the EM and to handle it safely. Dr. Jan van Lent of the Virology department of Wageningen UR gave us an introduction to EM with a small presentation. During this instruction he explained detailed the electron miscroscope itself, the preparation of good samples and how to operate the EM properly. Afterwards he showed the sample preparation in the wetlab. Preparation of the sample is critical to have a good resolution, during the course we learned how to do it. The sample must be thin, between 2 - 300nm, and stable in the electron microscope. This can be done by drying or cryo-freezing the sample. After drying or freezing, the sample is stained with a coating containing a heavy metal salt. This coating reflects the electron waves of the microscope, whereas the spots with the VLPs have no coating at all. The missing coating allows the electron waves to go through unhindered. The information of reflection and permeability is processed to an full image which shows the VLPs.
After the preparation of the samples Dr. van Lent explained the whole procedures of visualising a sample. After this last introduction we were able to analyse and investigate multiple samples ourselves.
At the end of the day we were able to use EM for our own experiments starting with sample preparation, handling of the microscope and proper visualization. After the course, the attending team members, are allowed and trained to use the EM without supervision.
Dynamic Light Scattering (DLS)
Another method to detect VLPs is dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasi-elastic light scattering. This technique is used to measure the size of particles. We applied this technique as a complimentary method next to electron microscopy.
When light travels through the sample and hits a particle, it will scatter in all directions. This scattered light is detected by the DLS. Due to Brownian motion, the particles moves randomly in the solution. These movements of the particles changes the distance of the partilces to the detector. This change of distance to the detector results in constructive or deconstructive interference of the scattered light. The change of intensity of the scattered light by the interference is related to the radius of the particle and can be calculated by the Stokes-Einstein equation.
We investigated various samples with the help of DLS. The main issue was that the sample was not pure or not concentrated enough. After optimization of the production and purification protocol we succeeded to detect VLPs. The main improvement was the implementation of FPLC, which filters out all aggregates and subunits, yielding pure VLP solutions.
DLS versus EM
Using both Electron Microscopy and Dynamic Light Scattering to detect our VLPs can be troublesome when the data of the EM and the data of the DLS are contradicting each other. To prevent this we compaired both methods with each other, to see if there are differences and explain those differences.
Sources:
EM
- Jan van Lent
- Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy - Leonard F. Pease, Daniel I. Lipin, De-Hao Tsai, Michael R. Zachariah, Linda H.L. Lua, Michael J. Tarlov, Anton P.J. Middelberg
DLS
- Remco Fokkink
- Light Scattering from Polymer Solutions and Nanoparticle Dispersions - Wolfgang Schartl
- Nanoparticle-Templated Assembly of Viral Protein Cages - Chao Chen, Marie-Christine Daniel, Zachary T. Quinkert, Mrinmoy De, Barry Stein, Valorie D. Bowman, Paul R. Chipman, Vincent M. Rotello, C. Cheng Kao, and Bogdan Dragnea
