You can put your "dogbowl dewar" under the microscope and focus it on
the surface of the liquid nitrogen. This works very well once you have
got the lighting right. You want to shine the light on the bottom of
the dewar so that it acts as a backlight for your loop. You can hold
the loop just above the surface of the liquid and it will still be very
cold, but much easier to see.
However, as has been mentioned there is no substitute for diffraction.
Transparency of the drop is neither necessary nor sufficient for good
crystal diffraction. Salty cryos do cool as amorphous solids, but often
have a "mottled" surface that is not optically clear, and
nano-crystalline cubic ice is optically clear, but still gives sharp ice
rings.
That said, some protein crystals diffract better in cubic ice than they
do in amorphous ice. The trick to the whole thing is avoiding strain in
the protein crystal lattice because if it strains as it cools then none
of your unit cells will be exactly the same size, and high-order Fourier
terms vanish. It is easy to imagine how strain could build up if your
solvent channels contract differently than the protein lattice. The
problem is that there is no way to know ahead of time how your protein
crystal lattice will interact with a particular cryo, so you have to
screen. I recommend taking a large VARIETY of cryo conditions to the
beamline for your first trip (don't just freeze everything in
glycerol). Mixed cryos are easier to glassify than single-component
cryos (the "confusion effect"). Don't forget oil, it works about half
the time (in my experience), and mind the level of liquid nitrogen in
the dewar or your cooling rate will not be reproducible (Warkentin et
al., JACr 2006). Or, better yet, if at all possible take uncooled
crystals to the beam and learn as you go. Protein crystals are a lot
like children, they are all special and unique and expect to be treated
that way, even after they are fully grown.
-James Holton
MAD Scientist
Claudia Scotti wrote:
Dear List,
Sorry for the probably silly question.
Any suggestions to test cryoconditions without X-rays or cryostream?
I'd need to freeze crystals before going to ESRF and I'm a bit
anxious. Is it enough to try to freeze the cryoconditions in liquid
nitrogen checking under the microscope (or by eye) or is this still risky?
Thanks,
Claudia
Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di
Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia
Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673
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