**Sample collection and data analysis for Single Crystal X-ray
diffraction studies at Beamline 11.3.1**

Allen Oliver and Sirine Fakra

Last updated, March 2005

These instructions assume some familiarity with goniometers and
diffractometers in general. Many of the instructions assume that you are
aware of how to operate a laboratory instrument, select and mount
crystals and how to center them. It is a guide to operating the D8
goniometer with the APEX2 software package.

1)  Examine your sample under the polarized microscope and mount it on a
    magnetic head. Use the Crystal Wand to support the magnetic head
    while your are mounting your sample.

2)  To mount and center your sample, connect to the **server** computer
    (black background with the word: "Server" displayed in the middle).
    To switch between the two Bruker PCs (server and client), double tap
    the "Scroll Lock" key on the keyboard.

3)  Make sure that BIS (Bruker Instrument Services) is running on the
    server before doing anything. It should be running at all times. If
    it is not, ask for assistance. Launch the APEX2-Server program
    (double click on the icon on the desktop).

4)  After the program launches select Instrument  Connection. Type in
    "**localhost**" in the text box and click on "Connect". You will
    have to wait while the computer communicates with the BIS software
    to set up the instrument. This usually takes 15 -- 30 seconds. You
    will be connected when the connection window disappears.

5)  Click on "*Simple Scans*" from the side bar. Wait for the window to
    appear in the main panel of the APEX2 window.

6)  To center your crystal drive the goniometer to omega = -180 and phi
    = 0 deg (enter the angle values in the appropriate text boxes on the
    right hand side of the window and click on the "Drive" button just
    below them). To rotate your crystal 180 deg. Click on the "Phi + 90"
    button twice then on the drive button. Center your crystal as well
    as you can in this orientation, then rotate the crystal to phi = -90
    or 90 deg by a single click of the "Phi +90" button. Center your
    crystal as well as you can in this orientation. Reiterate as
    necessary until you are satisfied that your sample is located along
    the phi-axis. Drive phi to 0 (zero) before continuing with the final
    height adjustment. With phi at zero, drive omega to -30 deg. And
    adjust as necessary. **DO NOT GO BEYOND OMEGA= -30 AS YOU WILL HAVE
    COLLISION.**

7)  Go to the Client computer to start your matrix and data collection
    (double tap the "Scroll Lock" key).

8)  Double click on the **Start Database** icon on the desktop. The
    database is necessary for APEX-2 to function. It keeps a record of
    the projects that have been run.

9)  Launch APEX2 from the desktop and login as "guest", the password is
    the same.

10) Connect to the server computer (Instrument  connection). The IP
    address of the server appears in the pop-up window, click on
    "**Connect**" and wait. Once the connection window disappears you
    are connected to BIS and are able to begin data collection.

11) Select the "***[Setup]{.underline}***" option (should be active) and
    click on the "*Describe*" module and enter the data as appropriate
    \[usually chemical formula, but crystal information (color, habit
    dimensions) can also be entered\]. After you have entered the data
    click on the lower of the two "**X**"\'s to close this module (**DO
    NOT** click on the upper-right-most "**X**" this will close the
    client and you will have to restart the whole process). It is quite
    important for the current version of APEX-2 to reduce the number of
    open windows, the scripting language sometimes gets confused. In
    addition, you will need to be patient as there are several portions
    within the program where there is a short wait for information to be
    loaded and transferred between the sever and client computers.

12) Select the "***[Collect]{.underline}***" option and select the
    "*Experiment*" module. You will be presented with a window that has
    a spread-sheet style layout describing how to collect the data. The
    field codes describe the various goniometer positions, collection
    times, angles that are scanned and so forth. The fields are similar
    to those found in SMART. This module is "tabbed". The currently
    active tab describes the data collection routine (similar to "Edit
    Hemisphere" in SMART). The second tab "[Monitor
    Experiment]{.underline}" shows the current frame as the data are
    collected.

13) Uncheck "**Correlate Frame**" and check "**Generate New Dark
    Images**". Next set the default collection time (typically a 1
    second frame is appropriate for screening samples). Set the
    "**Frame-Size**" to 512 x 512 (512 mode) or 1024 x 1024 (1k mode).
    Normally we run in 512 mode, but you may choose to run in 1k mode.
    Click on the "**Append Matrix Strategy**" button (lower-left corner
    of the main window) and wait while the data is loaded. Please be
    patient, it takes a short time for the software to compile and
    insert the data in the appropriate fields. When the runs have loaded
    there will be six lines of sample runs. The first four describe a
    "standard" matrix. **NOTE**: After you've run your matrix and
    analyzed it , change the filename from "matrix" to "Sample ID"
    (whatever you have chosen) before you begin collecting your full
    dataset. **DO NOT OVERWRITE FILE NAMES AS THE APEX2 SOFTWARE WILL
    HANG UP**. To prepare a matrix run, select the number "**5**" on the
    left-hand-most column and, while holding the "SHIFT" key down, click
    on the number "**8**" below it. Right-click on the "**Active**"
    field title and select "No". This will disable the last four blocks
    (which are the standard set-up for a full dataset; we don\'t need to
    run one of those right now). When you are ready, click on the
    "**Execute/Resume**" button on the lower-right-hand side of the
    window. This will start the data collection. There will be a pause
    while the server and client set-up the scans and data collection
    will commence. The pause also includes automatic collection of new
    dark frames. These will be collected at the start of each black of
    data. The window will automatically switch to the "[Monitor
    Experiment]{.underline}" tab, and your data will be displayed as it
    comes off the detector.

14) When your matrix runs have completed, select the "***[Evaluate
    Crystal]{.underline}***" option and launch the "*Determine Unit
    Cell*" module. Wait briefly for the computer to load the data and
    you see on-screen the first frame from your matrix run. The center
    of the screen displays the frame image. The right-hand side has the
    various functions necessary to "Harvest" (Threshold or Redetermine
    Unit Cell in SMART lingo) spots from the frames. The upper right
    hand corner displays the name of the first frame from the selection
    that you wish to use. The frame name format is:
    *filename_XX_YYYY.sfrm* XX = run/block number, YYYY = frame number.
    Hence you should see *filename_01_0001.sfrm* in the top corner.
    Below this is a number of frames that you wish to use for the matrix
    (it defaults to **20**, our standard matrix has **30**). Select
    "**Harvest Spots**" and you should see some green circles appear
    around reflections on the frame. Adjust the sliders to increase or
    reduce the number of reflections to be included (I/sigma(I)
    threshold). You are also able to select a frame to view with the
    right-hand slider. Typically 7 marks on the slider appears to give
    sufficient data to obtain a unit cell. Click on "**OK**" to store
    the reflections. Change the block number to the next in the sequence
    and repeat until done.

15) Select "**Index**" to begin indexing reflections. It is advisable to
    check the "Spots Must Span Images" box and set the resolution limit
    to something reasonable depending on the observed diffraction
    pattern f your sample (0.8 A is good for many samples). Adjust
    sliders to your hearts content and click on the "**Index**" button
    in the pop-up window. This will then attempt to index the
    reflections and give you a reduced unit cell. If the routine is
    unable to determine a unit cell it is likely that your crystal is
    not very good. Try indexing again with tighter tolerances on the
    data. Click "**OK**" to continue.

16) Choose the "**Refine**" button to refine your unit cell data and to
    select a Bravais lattice. Initially the least-squares analysis of
    your matrix data will be quite intolerant (the slider will be very
    low down). This is due to an algorithm behind the refinement
    program. The number of reflections included in the refinement is in
    the middle of the window. Usually one "click" on the slider will
    introduce a significantly larger number of reflections to improve
    the unit cell. The upper panels display parameters relating to the
    instrument and crystal position. The first two check boxes (crystal
    to detector distance and beam center X and Y) should be unchecked
    and the remaining three checked. Click on the "**Refine**" button to
    refine the cell data once.

17) Now select the "**Bravais**" button. The program will give you its
    best guess as to the lattice type and Laue group of the cell. Use
    your best judgement to decide if the program is correct. If you
    think it is otherwise, select the lattice/Laue group that you think
    is correct. Once you are satisfied click on "**OK**" to return to
    the cell refinement window. If at any stage you think you may have
    made a mistake, click on the "**Cancel**" button. This will quit the
    refinement routine and not save the refined parameters. If you click
    on "**OK**" it will save the newly refined parameters.

18) You will now start a quick iterative cycle of cell refinement to
    improve the detector, goniometer and crystal position parameters.
    With the newly selected cell, click on "**Refine**". Check the
    crystal to detector distance and beam center boxes and uncheck the
    detector roll, pitch and yaw parameter. Refine these. Step through
    the other options (crystal position and goniometer zeroes) in a
    similar fashion. IF your cell refinement fails, the software will
    apparently lock up and/or you will see a pink panel appear in one or
    more of the parameter boxes. Simply click on the "**Cancel**" button
    to quit the cell refinement. Save your project, quit APEX-2 and then
    restart it. You will be able to come back to where you were. This is
    currently the only way to clear the "Egg-timer" that will appear.

19) Cycle through several iterations of cell refinement with each of the
    detector/goniometer and crystal parameters until finally you can
    have all of the parameters refined together. NOTE: Keep an eye on
    the frame window to the left, it will show the predicted spot
    locations. You can decide if the cell is suitable and refining well
    visually. That is, watch the green circles and make sure that they
    over-lap your reflections. You can scroll through frames and blocks
    of data by using the arrow keys displayed at the top of the frame
    window.

20) Once you are satisfied that you have obtained the best cell and
    orientation matrix that you are able to, you can save the P4P file
    by selecting File  Export  P4P File. You will be prompted for an
    output file name (it defaults to the project name). Click on "OK" to
    save the P4P file (of potential use for post facto data analysis).

21) You are now ready to begin data collection. Close the "*Determine
    Unit Cell*" window (click on the X) and return to the
    "***[Collect]{.underline}***"  "*Experiment*" window, "[Setup
    Experiment]{.underline}" tab. You need to turn off the matrix
    collection routine and enable the data collection routine. To do
    this, click on the numeral "**1**" on the left-hand side. While
    holding down the "SHIFT" key, click on the numeral "**4**". This
    will highlight the first four blocks of data. Right-click on the
    "Active" field name and select "No". Repeat this for the blocks
    **5** through **8**, back make them active instead ("Yes").

22) Click on the "Validate" button. This will determine if the set of
    runs is viable or if it has potential collisions. This is imperative
    to do if you modify the standard set of runs in any way.

23) Once you are ready to start your data collection, uncheck the
    "**Generate new Dark Frames**" check-box, this will allow the Izero
    value to be recorded in the frame header. Then click on the
    "**Execute/Resume**" button and data collection will begin. The
    computer will switch over to the "[Monitor Experiment]{.underline}"
    tab and display the frames as they are taken.

24) Once your data has collected you will need to integrate. Select
    "***[Integrate]{.underline}***"  "*Integrate Images*". The window
    will open with a spread-sheet feel to it. To import the frames that
    you collected click on the "**Import Runs from Experiment**" button.
    You will probably need to set the resolution to a lower limit than
    the software decides (text box in upper right-hand corner). You will
    need to choose the appropriate space group for the crystal system
    that you have (the default space groups for each setting are picked
    out with an asterisk next to them)

25) Click on the "**Refinement Options**" button; you need to set the
    box size to 1.5 X 1.5 X 1.5 and uncheck the Enable Box Size
    Refinement. Also set the "*Frequency (Images)*" number to 100
    frames. You also need to set the local refinement to refine crystal
    translations (under the left-hand *Refinement Parameters* window).
    When you are satisfied that these are correct, click on "**OK**" to
    accept these parameters.

26) In the "**Integration Options**" you need to decide whether or not
    you wish to correct for beam decay. If you do you need to check the
    "*Enable Beam Monitor Normalization*" box. Under "**More Options**"
    you will need to unselect "*Multi-wire Reference Correction*". Then
    click on "**OK**" to accept these changes.

27) Once you are ready, click on the "**Start Integration**" button.
    This will launch SAINT in the background and SAINT Chart in the
    foreground. SAINT Chart is a graphic display of the integration
    progress. Watch it for any unusual features. It is simply a
    graphical representation of the usual numeric SAINT screen output.
    Once the integration has finished, click on the "X" at the top right
    of the window to close the SAINT Chart window. Close the Integrate
    window by clicking on the lower of the two "X" in the top right.

28) Once the integration has completed you will need to scale and apply
    absorption corrections. APEX-2 provides a GUI to the SADABS program.
    Click on "***[Scale]{.underline}***"  "*Scale*". This will open the
    GUI for SADABS.

29) Check the "Additional Spherical Absorption Correction". For routine
    samples, given their frequently small size, a mu\*r of 0.01 or 0.02
    should be sufficient. Uncheck the Lambda\\2 Correction. The beamline
    has light polarized in a different fashion to laboratory sources.
    Next click on the "folder" icon at the top right (next to the text
    box named: Base"). This will open an additional window, select the
    first blocks RAW filename, check the "**Open all with Base**" box
    and click on "**OK**". Uncheck the "**Merged Batches**" box; this
    will then use all of the separate runs for absorption correction.
    Finally click on "**Next**" to go to the "*Parameter Refinement*"
    tab.

30) The "*Parameter Refinement*" tab shows data about your unit cell (as
    obtained from the final cell refinement, the wavelength of the
    radiation, total number of reflections, number of unique reflections
    etc ... It is simply a graphical alternative to the SADABS
    interface.

31) Depending on your usual lab practices, the mean I/sig(I) threshold
    should be set to 3 or 5, and Absorption Type to "*Medium Absorber*"
    (this applies a \[6, 3\] spherical harmonic correction to the data,
    more or less unusually shaped crystals should have a strong \[8, 5\]
    or weak absorber \[4, 1\] applied as necessary). You can also modify
    the high angle cut-off if your data were integrated to too high an
    angle (*filename*m.ls output will reveal this). When you are ready,
    click on "**Refine**" to apply the absorption correction. The graph
    on the lower left will show the applied correction and R(int) for
    the data. It should refine out to a flat value. To move to the final
    analysis of the data click on "**Next**".

32) You will now enter the "*Error Model*" portion of the program. The
    defaults are acceptable. Ignore the graphic displays when they come
    up. They are just graphical interpretations of the incident and
    diffracted scale factors, R(int)'s for each block of data etc. The
    titles are self-explanatory. When you are ready to determine outlier
    rejection etc, click on "**Determine Error Model**". After this is
    completed, you may wish to re-refine the absorption correction
    parameters with new g-values. This is not normally needed. Finally
    click on "**Finish**" to write out the HKL file and prepare the
    usual SADABS graphs. View the graphs to make sure that nothing
    untoward has happened to your data (if it has, go back and
    re-examine the frames for any obvious errors and re-determine the
    cell/re-integrate if necessary. To quit the scaling/SADABS interface
    click on the "**Exit AXScale**" button.

33) The penultimate function is the "***[Examine Data]{.underline}***" 
    "*Space Group Determination*" (XPREP). This is precisely the same as
    the laboratory version. You should be familiar with its
    functionality. If not, go to the CHEXRAY web pages and help files
    found there-in.
    ([http://xray.cchem.berkeley.edu](http://xray.cchem.berkeley.edu/)).
    NOTE: You will need to change the wavelength of the radiation used
    in the experiment (at the cell contents portion of the program). We
    typically use 0.775 Å radiation (16 keV).

34) Ultimately you will solve your structure. You will need to solve and
    refine your structure using SHELX, as it is capable of incorporating
    scattering factors of elements for radiation other than molybdenum,
    copper etc. You will need to use the program CROMER to determine the
    scattering factors for the elements present in your sample at the
    wavelength used (usually lambda = 0.775 Å).