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Particle picking guidelines
Home > Picking particles > Particle picking guidelines
The power of EM lies within the fact that you will see your sample on your grid no matter if the sample is well preserved OR falling apart. This means that you need to have a discerning eye in order to make sure you are picking real particles, and not simply noise/aggregates/degraded molecules.
Checklist for imaging a new sample:
- Does the diameter of the single particle agree with expected dimensions?
- Is the sample fully embedded in stain?
- Is there a predominant species on the grid? Is the grid homogeneous?
- Are there aggregates and how often do they occur?
The guiding principle through single particle EM is asking yourself this important question:
Do the dimensions of the particles in my image agree with the expected size, based upon its molecular weight?
This is critical when preparing grids of a NEW sample. To help you, here are the diameters (in Angstroms) of a number of samples:
80S eukaryotic ribosome (4,000 kDa): 250 Angstroms
Nucleosome (~200 kDa) - 110 Angstroms
gamma-secretase (~170 kDa) - 180 Angstroms
In order to be able to interpret negatively stained particles, there must be appear to be a homogenous layer of stain/ice around the particles.
For negatively stained samples, you should not see any dark halos around single particles. If you see this, the particle is likely only partially embedded in the stain, which means that you are not seeing the full complex.
For instance, the following image will highlight different aspects of what you might see on your grids.
Below is an image showing partially embedded particles on the left (a) and fully embedded particles on the right (b).
In general, you do not want particles that have a dark halo like in (a). To avoid these particles, simply find a region of your grid that has deeper stain, which is usually possible.
In addition to finding regions of 'deep stain' on you grid, you must be able to identify: 1) if your complex is falling apart and 2) if the sample is aggregating.
A sample falling apart on the grid is likely due to the hydrophobic force exerted by the air/water interface, which likely occurs when the stain is too thin (and can't 'protect' the sample from the air/water interface). You can notice this happening by seeing small dots decorating the background of image, where the diameter of these dots is much smaller than the expected size of your sample.
An example of this is shown here for a grid of the 26S proteasome, which is made up of a core peptidase and an auxiliary 'regulatory particle' that caps the peptidase. There are > 15 subunits within this complex, so when there is thin stain, you can see images like this:
Where you see mostly smaller objects than that of the large 26S proteasome. This means either that the sample fell apart during purification OR it fell apart on the grid during preparation.
A sample aggregating is manifested as large clumps of sample sticking together on your grid. While you may be able to discern your sample in the aggregate, these are NOT able to be used because analysis routines would not be able to differentiate between the overlapping particle density.
Aggregates can be clearly seen in the above image of the proteasome, which are the large dark clumps of protein.
A good rule of thumb: If aggregates comprise (on average) >10% of image real estate, you should try to investigate possible causes of the aggregation.
Source:
Images taken from: Ohi et al. (2004) Biol. Proceed. Online