/Interpreting Results.png "The use of the UV-TPEF channel which provides a clear assessment of each drop")
Sitting drop examples
In general, the SHG imaging channel will provide the ability to quickly determine which wells contain protein crystals. However, because all samples are different, it is possible to have higher backgrounds than expected and signal from dust or salts resulting in false positives. The use of UV-TPEF provides the ability to rule out these false positives and to definitively determine whether or not regions of interest are protein crystals.
Tops of plates and sealing tape may generate significant amounts of SHG and fluorescence. If the laser is focused on these regions, then one may expect strong background interference from these materials. However, the depth of focus of the beam is small enough that when the laser is focused in the drop, there should be no out of plane contributions from the plates and tape.
Some salts that produce noncentrosymmetric crystals (either chiral or achiral molecules) will produce SHG making it difficult to distinguish between salt and protein crystals. The UV-TPEF channel is used to discern whether the crystal is of proteinaceous material. Those crystals that contain tryptophan will fluoresce when imaged with the 532 nm beam.
If both the SHG and UV-TPEF channels show similar contrast then you can be certain that your sample is a protein crystal (unless you have fluorescent small molecules present). Note that all protein material, whether crystalline or not, will show up on the UV-TPEF channel, so it is important to compare to the SHG channel to determine if it is crystalline.
The use of the UV-TPEF channel provides a clear assessment of each drop which can be seen in the figure below. In both rows (a) and (b) contrast is observed in both the UV-TPEF and SHG channel indicating protein crystals. The well imaged in row (c) results in fluorescence but no SHG, indicating precipitated/aggregated protein. Salt crystals can also produce SHG as shown in row (d) but result in no fluorescence making it easy to distinguish between salt and protein crystals.
Sitting drop examples
|
|
| RIC-V33R116 |