UV FAQs
If your question does not appear below, please email support@formulatrix.com and we will respond within 24 hours, Monday through Friday.
► What plates and cover materials are compatible with UV?
Answer: Plates vary, and can even vary within a brand. The most confident answer we can provide is that polyolefin works fantastically, and polystyrene can vary from not good to OK.
► What is the smallest protein crystal that the UV camera can pick up?
Answer: This answer is dependent upon your plate. On a perfect plate, the answer is roughly 5-7 μm for a continuous zoom UV.
► Why do some proteins fluoresce more than others?
Answer: Multiple causes. The signal is directly proportional to the concentration of tryptophan. Plate media can dampen the signal, or introduce noise that when compensated for leaves the impression of less fluorescence, which is why plate masking is important for UV imaging. Also, the drop fluid itself could be absorbing inbound excitation UV or outbound emission UV.
► What concentration of tryptophan is needed to make the protein crystal fluoresce under UV?
Answer: Technically, any tryptophan will fluoresce, at any concentration. As far as what concentration threshold is needed in order for the florescence to translate to the image captures — the answer varies with exposure time, ambient light, gain setting, plate media interference, etc.
► Can we adjust the brightness of the UV LED?
Answer: No. The LED is already at it's maximum without burning out as it is. Too much LED will cook your protein samples.
► Do glass cover slides degrade the fluorescence?
Answer: Some interfere, some do not. The best way to check is with a spectrophotometer.
► Can you get false negatives or false positives in terms of determining if a crystal is a protein or not?
Answer: False Positives: Some other things fluoresce — especially if they have aromatic rings in their chemical structure. This includes some detergents, some plastics (polystyrene), skin cells (trytophan, in that case), and even the artificial sweetener saccharin. Generic room dust contains an impressive amount of skin, so dirty plates might get some spots on them.
False Negatives: Tiny crystals beyond what we can resolve will be difficult to focus on, or difficult to distinguish - these could be considered false negatives. Actual false negatives would only occur one of three ways:
- UV doesn't make it to the crystal. This could be caused by media absorption, or (less likely) solution absorption. UV might not penetrate into a very, very deep well, which can happen in 5 mL drops.
- UV makes it to the crystal, is absorbed, but the protein does not fluoresce (no tryptophan, damaged/bleached tryptophan).
- The fluoresced UV does not make it to the camera (media absorption or solution absorption again).
All three of these scenarios are unlikely, given carefully chosen media and reasonable drop sizes.
► Where do you place the UV lens? Does it image from the top of the plate or from the bottom? If from the top, then we will have to put the glass sandwich plates upside down. Does it affect the imager operation?
Answer: The UV lens is above the plate. Putting the plates in upside down should be fine — you’ll just need to switch the barcode to the other side. You may want to try this in your system to make sure there are no unexpected consequences.