Translate it by 13 microns. And use enough attenuation to get 180
degrees at each position.
The track length of photoelectrons from 1 A X-rays in water, protein,
plastic, and other materials with density close to 1 g/cm^3 and atomic
numbers close to 7 is about 3 microns (Cole, Rad. Res. 1969). This
defines the effective maximum "range" of the radiolytic chemistry. So,
10+3 = 13 microns from center-to-center if you want to avoid the damage
of the last shot.
That said, if you blast the living daylights out of one spot you will
eventually be able to see it grow in size, and the uneven expansion
produces stress that can propagate into the unilluminated areas of your
sample. It stands to reason that stress is not good for diffraction, so
you could consider this "dose contrast" effect as a mechanism of damage
spreading. Nevertheless, it has been shown that at "moderate" doses
(spots fading noticeably, but not disappearing entirely) properly
accounting for the dose to the illuminated volume under different dose
contrast situations leads to similar decay curves (Zeldin et al 2013),
indicating that dose itself is a lot more important than dose contrast.
Perhaps the main reason why "damage spreading" is still not all that
well understood is because it is really really hard to produce an X-ray
beam with edges sharp enough to study it! This is because all X-ray
beams have some divergence (aka "crossfire"), and it is generally unwise
to put a collimator inside the cryo stream. At 1 cm from the sample,
even with the relatively low divergence of 100 microRadian (0.006 deg)
the X-ray beam will be 1 micron bigger at the sample than it was at the
collimator, blurring at the edges. You can reduce the divergence, but
that will cost you flux. Balancing all these considerations for making
a small beam generally results in a Gaussian shape, so I'm willing to
bet your 10 micron beam is Gaussian. For any Gaussian beam half of the
incident photons fall outside the full-width-at-half-max (FWHM) contour
level generally quoted to define the "size" of the beam. No doubt a lot
of people who think they are seeing damage "spreading" into regions
outside the beam-box are actually seeing nothing more than damage caused
by the tails of the main beam itself. Without collimation, these tails
formally extend to infinity, so the question of how far to translate
becomes not one of how to completely avoid damage, but how much damage
you are willing to put up with. Is 10% okay? 5%? 20%? Remember, that
even your first shot on a "fresh" part of the crystal is not going to be
damage-free because damage is going on during each exposure, including
the first one! (unless, of course, you are using an XFEL).
You can do a lot of math trying to optimize diffracted photons vs damage
(see Zeldin et al. 2013), but at the end of it all you find that the
best way to utilize a given volume of "good" scattering matter is to use
a beam that evenly illuminates that volume. This is because any bit of
"good stuff" that never sees beam is wasted, and over-exposing one bit
over another doesn't gain you anything. You also don't want to shoot
things that are not "good stuff" because that corrupts your data with
background and/or unwanted spots. Unfortunately, adjusting beam size to
match each crystal shape exactly is a major engineering challenge and
even if you could do this the sample has to rotate, making avoiding at
least some "unwanted material" impossible. So, in reality, your beam
size tends to be fixed and you must "paint" with it on the canvas of
your large, rotating crystal. You can run simulations of such
strategies at http://www.raddo.se/, and there are some tricks like
off-setting the beam from the rotation axis to better approach even
illumination, but in the end you cannot escape the even-illumination
optimum. To that end, a train of Gaussian profiles separated by their
FWHM forms a profile that is "flat" on top to within 10%. So, once
again, since the damage from a 10 micron beam is 16 microns wide, a
translation of 13 microns per "wedge" is a decent compromise. Hence my
recommendation above.
The next, question, of course, is how many shots you can get per
"wedge". I have written a web jiffy for answering questions like this:
http://bl831.als.lbl.gov/xtallife.html
Since you mention metals in your crystal, I'm going to assume this is a
metalloprotein, and metalloprotein active sites can be particularly
dose-sensitive. For example the water-splitting complex in
Photosystem-II has been shown to decay with a half dose of 500 kGy
(Yano, 2004), but the standing world-record is myoglobin, reducing half
its iron with only 20 kGy (Denisov, 2007). Taking 500 kGy as your dose
limit, and assuming you are using 1 A X-rays, I can type in the
parameters you describe into the above web page and I get ... an error
message. This is because the beam you are using delivers 5 MGy/s, so
your first 0.1 s exposure has already hit the dose limit. The program
responds to this situation with attenuation, which is an "okay"
solution. With 91% attenuation (9e10 photon/s) you should be able to
just get a full 180-degree wedge with an average dose on the order of
500 kGy. And yes, you do need to do a full 180 to hit every part of a
crystal that is bigger than the beam in both the "vertical" and
"thickness" directions. Hitting it all evenly is another story. In
general, it is difficult to fully utilize a large crystal with a small
beam without over-burning the core. You've got some "painting" to do.
Translating the crystal helps, but if you don't get 180 degrees at each
position then you've got a double helix of unexposed crystal left over
at the end, making your "end game" a bit complicated.
For situations with such wildly miss-matched beam and crystal sizes, I
recommend running RADDOSE-3D. When I do that, I find that flux = 1.6e10
photons/s (aka 98.4% attenuation) is required to get a
diffraction-weighted-dose (DWD) of 500 kGy in a 180 deg sweep. The
"core" is over-burnt at 9.66 MGy, but it is only 0.25% of the total
exposed volume. This volume, however, is ALWAYS in the beam, so it
"counts" more than the rest. This is what the DWD statistic is all
about. Fundamentally, it assumes that the consequences of dose are
linear, so you can weight them by the time they spend in the beam. This
makes sense when the dose contrast is relatively low, but how much a
diffraction pattern from a 1:20 ratio mixture of crystal regions at 9.66
MGy and 0.018 MGy looks like that from the same volume evenly-cooked to
0.5 MGy is still a very good question.
Yes, it gets complicated, doesn't it? This is why I generally recommend
trying to use a beam that matches your crystal size.
-James Holton
MAD Scientist
On 12/29/2014 2:17 AM, Mohamed Noor wrote:
Dear all
In a metal-containing crystal of (say) 200 um x 200 um, and a beam size of 10
um x 10 um, how far will I need to move away from an irradiated part to a fresh
part to obtain an undamaged dataset?
Exposure conditions: 100 % transmission at 10^12 ph/s, 0.1 s exposure, fine
sliced at 0.1 degree/frame with a total 180 degrees.
Obviously it will be crystal dependent but I would like to have a rule of
thumb. I could use fresh crystals altogether, but not all crystals diffract
well unfortunately.
Thanks.
Mohamed
