ROCK IMAGER

Not all crystallography consumables are compatible with ultraviolet light. Cover media can either block the excitation light (absorb UV radiation in the 250-320nm band) or it can fluoresce in the same band that tryptophan does, creating a high level of background noise (this is a process commonly called autofluorescence). Crystallization plates can also autofluoresce, increasing unwanted noise. The challenge of absorption is not a problem in crystallization plates for we have positioned the UV light source and camera of our imagers above the plates.

[The term ´autofluorescence´ is a bit of a misnomer. Typically, you've done something to make your target fluoresce, and this noise-contributing fluorescence happens automatically, hence autofluorescence. Here, we're using the intrinsic fluorescence of tryptophan, which is the exception rather than the rule in fluorescence microscopy. Our desired signal is, in a sense, already autofluorescence. In this document though, we use the term autofluorescence to refer to undesired noise from the cover media or plate.]

To recap, there are three consumable-related problems that could reduce the signal-to-noise ratio while imaging using UV light (listed in the order of what seems most common/serious):

• • Crystallization plate could autofluoresce, increasing noise (common in polystyrene plates).
  • Cover media could absorb some/all of the UV excitation light, reducing the signal strength.

[Note: This can be partially recovered with longer exposure times]

• Cover media could autofluoresce, increasing noise (common in glass cover slides).

Notes:

• Plates and cover media that offer "low birefringence" seem to also have low autofluorescence. This is the best rule of thumb we´ve found so far.
• At least twice, media manufacturers have changed formulations for some nonrelated reason (hydrophilicity, optical properties, etc.) and inadvertently decreased UV performance. The two cases we´ve noted were for TTP LabTech´s ViewDrop sheets and Hampton Research´s glass cover slides. We have notified both manufacturers, and they are interested in addressing their respective problems.