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ROCK IMAGER User's Guide | Chapter 9: Using RI 2, FRAP Benchtop & SONICC Benchtop
4. In the
Experiment
contextual tab set, click the
View
tab, and then click the
Imaging
Settings
button in the
Info
group.
5. The
Imaging Settings
panel will appear next to the
Drop
tab. Look for “End-State image
offsets” and “Post-Bleach image offsets”. Offset values should range between 5-10 µm.
If the offset value differences are
greater than ~30 µm, it may point to a
problem with the stage or the image
alignment algorithm. Email
assistance.
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 non-centrosymmetric 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.
Note:
FRAP alerts you when there is an image alignment issue on the
pre-bleach and end-state images so that you can be aware of any possible
issues. If you see an image with an error message and a score of -1, it means
that the software algorithm was unable to align the pre-bleach and end-state
images within the specified offset tolerance.
FRAP Offset Values in ROCK MAKER
Interpreting SONICC Results
