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UV Imaging of Crystallisation Experiments

Viewing UV images at C3

Jump to About UV Imaging

  • UV imaging occurs on every 5th inspection, coloured red below
  • Sitting drop plates are imaged on days 0 (same day as setup), 1, 3, 5, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70
  • LCP plates are imaged on days 0 (same day as setup), 1, 2, 3, 4, 5, 9, 13, 16, 20, 24, 27

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).

About UV Imaging

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:

  • If the protein has no tryptophan residues, the UV fluorescence will be minimal.
  • Most – but not all – salts do not fluoresce when illuminated UV light, so just because you see a crystal in a UV image does not guarantee that it is a protein crystal.
  • Heavy protein precipitate will also fluoresce, for the same reason that crystals glow brightly – it is a concentrated region of protein.
  • Protein can concentrate at an air/liquid interface
  • Some cofactors (haem, for example) appear to quench the UV fluorescence, and some of the chemicals used to crystallise proteins also attenuate the UV fluorescence – we have seen this with MPD, amongst other chemicals.

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).