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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.
This answer is dependent upon your plate. On a perfect plate, the answer is roughly 5-7 μm for a continuous zoom UV.
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.
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.
No. The LED is already at it's maximum without burning out as it is. Too much LED will cook your protein samples.
Some interfere, some do not. The best way to check is with a spectrophotometer.
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.
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.
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