Not all crystallography consumables are compatible with ultraviolet light. Cover media can either block the excitation light (absorb UV radiation in the 250-320 nm 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 autofluoresceThe 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., 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.
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 (This can be partially recovered with longer exposure times).
Cover media could autofluoresce, increasing noise (common in glass cover slides).
▶ 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 non-related 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.
Formulatrix has tested several cover medias and plates. For the most up to date information, please see the online Formulatrix Plate Guide.
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