Jump to About UV Imaging
These can be viewed in See3, look for inspections with 2 sub-inspections (red, below)
If you are on an inspection with two sub-inspections, then hitting the ‘split screen’ button (red, below) will display both the visible image and the UV image side by side.
Alternatively when viewing a single drop with subinspections, you can toggle to UV images by changing Mono 2mm to UV 2mm (red, below). For further information see the C3 help pdf (blue, below).
Most proteins contain tryptophan, which when excited by UV light will fluoresce at between 300-350nm (it is solvatochromic). Given that protein crystals are concentrated areas of protein, they should glow brightly when illuminated with UV light. The UV imaging thus allows one to locate protein crystals easily, and to validate that you’ve grown a protein crystal and not a misleading salt crystal.
Obvious visual and UV imaging of protein crystals
‘Invisible’ protein crystals found using UV
Salt crystals and some localized precipitation
In C3, both of our imaging systems are UV capable, and we image with UV illumination three times during the regular (visible) inspection schedule, once on inspection 5, and again on inspections 10 and 15. This is approximately after one week, then after one month and finally on the last scheduled inspection. Of course there are caveats to the UV imaging:
Look at the visible timecourse of your droplet as well as at the UV inspections to really interpret what you see. Protein can precipitate at any air/liquid interface, and bubbles created during robotic set up are no exception. Over time, the bubbles shrink, but the shadow of the bubbles can be seen in a ghost of protein skin which remains. This crumpled skin can fluoresce and glow just like crystals.
Insulin crystals which will not fluoresce in UV light
The images highlighted in green contain nitrates which quench UV fluorescence
READ MORE: Viewing Images
Reference: Desbois, S.; Seabrook, S. A.; Newman, J., Some practical guidelines for UV imaging in the protein crystallization laboratory. Acta Crystallographica Section F 2013, 69 (2).