A sitting drop with crystals
Since proteins are so exceedingly difficult to crystallize, the goal is to grow the protein crystals just large enough to allow for X-ray diffraction patterns good enough for structure solution.
Practically speaking, this means that protein crystals need to be detected with a high resolution microscope. Ideally, this microscope should be optimized for protein drop imaging. This affects optics, but in particular also the light source. Since we are imaging a drop of liquid, the edges of the drop can be obscured due to light diffraction at the edges of the drop. Unfortunately there are cases where crystals are missed because conventional light sources fail to illuminated these regions adequately. The image above is a good example to illustrate this effect
Needle crystals imaged using visible light. Click on image for a high resolution view.
Compare this to a drop imaged with specialized optics and properly illumination (right image). It is worth to look at this image at full resolution (just click on the image). You can see needle crystals alongside precipitates. Oftentimes, these precipitates can be dense enough to the point where crystal detection becomes very difficult if not impossible. This is a severe problem e.g. in membrane crystallization where the entire drop can be opaque to the point where visible light microscopy needs to be complemented with other methods, in particular UV microscopy.
Same needle crystals imaged using UV light at 200ms exposure. Click on image for a high resolution view.
In UV microscopy one exploits the fact that most proteins contain either tryptophane or tyrosine (or both) that can be excited using UV light. As a consequence, proteins fluoresce when inspected under an UV microscope, increasing vastly the chance to detect small protein crystals in complex drops. The main drawback of UV microscopy for protein crystallization is that if the UV light source is too intense or the exposure time too long, the protein crystal can deteriorate to the point where structure determination from these crystals becomes impossible. Therefore, special attention should be give to picking the right UV light source for imaging of protein crystals. But if it’s done right, it is just fascinating seeing protein crystals light up!
UV imaging at the JCSG.
More posts on crystal detection.
Click here to learn more about crystal detection at Rigaku.com.
