Note the density of air is approximately 1000 times less than a protein crystal. The total scatter for a beam going through a 50 micron thick crystal will be similar to that from 50mm air. Most beamlines will have a path length less than this but nevertheless the air scatter will be significant with small crystals. In principle, with smaller beams one can have smaller beamstops nearer the sample thus reducing the path length through the air.
Colin -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Richard Gillilan Sent: 26 November 2007 16:24 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] To bathe or not to bathe. > I just noticed this thread. I should make a few comments. We regularly provide microbeam with and without Helium here at MacCHESS. Yes, there are cases in which microbeam can give you good diffraction on large crystals when a larger beam cannot. Just last week we had a user group collecting on column-shaped crystals about 40 microns wide by 200-300 microns long. When exposed using a 150 micron diameter beam, the diffraction patterns were a mess, producing multiple lattices. When they exposed the crystals using a 20 micron beam, they were able to find enough "sweet spots" with single lattice to give good results. I can only offer anecdotes at this time, but, based on user experiences so far, this is not uncommon at all. I don't usually think of microbeam alone as a significant reduction in background except that one avoids hitting solvent (which is a major source of scatter!). While the beam scatters less air due to its small size, microbeams also can have much increased flux which compensates for the smaller size ... so I don't think there will be less air scatter unless you are just using an aperture with no flux gain. Regarding helium: we regularly use helium at 95K combined with a helium enclosure. The setup is awkward for manual sample mounting, but quite convenient for automounting. The main advantage here is that direct beam scattering with air is nearly eliminated. How important is this effect? It depends upon how much free path there is between the end of the optic (slits, etc) and the beamstop. It also depends upon how small your sample is and how strongly it diffracts. In a carefully-controlled experiment using the identical crystal and orientation range with both nitrogen and helium, I saw a signal-to-noise improvement by over a factor of 3 in the 2.5 Angstrom range and lower. At higher resolution the benefit decreases, but still looks no worse than a factor of 2. This is with a 50 micron crystal illuminated with an 18 micron beam. I am currently working on guidelines for when helium and microbeam are necessary (based on both simulations and explicit measurements). At the present time, my feeling is that crystals below 50 micron can certainly make the extra hassle worthwhile. It really depends upon how badly you want that extra resolution. In the case above, it pushed the resolution from above to below the 2.0 Angstrom mark based on I/SIG. As for radiation damage, I do think it is important that a beam intensity profile be as flat at the top as possible (not a sharp hotspot) .. so defocusing a little may be useful. This is not usually a factor that regular users have any control over. I don't have any hard data on this. Richard Gillilan MacCHESS <DIV><FONT size="1" color="gray">This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom </FONT></DIV>
