Our last post about UV microscopy was all about optical resolution. We used protein needle crystals to get an idea what smallest size protein crystal would be that that we could resolve with our new imager. Of course we were incredible proud about the results: needle diameters smaller that 2.5 μm! In the meanwhile we revisited this particular drop – only to find that the resolution limit is actually 2.0 μm or better – but that aside, UV microscopy in protein crystallization is not all about resolution of small nuclei, but also about detection of larger crystals obscured by the actual drop.
Small crystals under heavy precipitate
There are three main scenarios in protein crystallization where visual microscopy fails, independently of the resolution of an optical microscopy: Heavy precipitates, salt formation, and otherwise opaque screens, such as the ones found in membrane crystallization.
The image above shows the effect of heavy precipitate. The visual image is complex to a point where small protein crystals cannot be identified. Under UV light the situation is completely different since the fluorescence of the precipitate is significantly less intense than from the actual protein crystals.
The next picture shows a drop where a large salt crystal obscures small concanavalin A protein crystals. Again, under visible light it is impossible to detect the small protein crystals that are easily distinguishable under UV light.
Tiny protein crystals hidden under crystallized salt - easily detectable with UV
Finally, UV microscopy is great news for membrane protein crystallization. Below are visible and UV images for a thermatoga screen – I am not saying that you can’t see the protein crystals at all under visible light at all, but what a help the UV image is!
Crystal detection in opaque membrane screens
I’d like to thank my colleague Pierre LeMagueres for sharing all above images with us!
