Sample Tab

The Sample Tab allows the user to prepare the sample for data collection: The user can change the sample camera zoom and adjust the sample position, change the beam size , mount and dismount additional samples with the SAM robot, remove ice, and anneal the crystal.

Mounting the Crystal

Manual Mounting

  • General directions for mounting a crystal in a cryoloop can be found in this slide show.
  • Move the detector and beamstop away from the sample for easy mounting. On BL12-2 make sure that the on-axis camera back-light is removed, by selecting the "On-Axis" Sample camera video and clicking the Remove button under "Sample Lights Control". Please do not attempt to adjust the beam stop manually. If the beamstop is bent out of the beam or broken it will expose the detector to direct beam and damage it.
  • Practice mounting a pin with a blank loop on the goniometer to make sure your system is compatible with our coaxial cold stream. Contact Staff if you run in to any problems.
  • Check that the cold stream is aligned correctly (nozzle to sample distance should be ~10mm).
  • Mount your sample on the goniometer head.
  • Move the beamstop back into place.
  • Use the resolution predictor to calculate the optimal beamstop distance.

If you will be using the Stanford Automated Mounting (SAM) robot for mounting your crystals, see either the Sample tab or the Screening tab for further instructions.

Mounting with the Robot Interface

  • This interface on the Sample tab may be used to mount crystals one at a time using the Stanford Automated Mounting (SAM) robot. You must have loaded your cassette(s) into the robot dewar inside the hutch. Using this interface does not necessarily require you to have uploaded a spreadsheet containing information about your cassettes (see Screening tab for details). The only information that is required is the location of your cassettes (left, right or middle). This interface can be very useful if you only have a few crystals in a cassette to collect, or if you have screened a cassette using the Screening Tab and have decided which crystals you want to go back to and collect.

The procedure for mounting and dismounting a crystal is:

  • choose the correct position of your cassette (left, right or middle) by selecting the correct tab above the table.

  • the interface will probably return a (u) next to the position indicating that the robot does not know whether a cassette actually exists in that position or not. If you have previously used the Screening Tab interface to screen this cassette, then you may see (1) next to the position. Either way you may continue.
  • you may also see that all the crystal locations in the table show as grey circles. This means that the robot does not know whether a pin exists in that location or not. Again, if you have used the Screening Tab to screen your cassette, there may be a combination of different colored circles with green meaning that there is a pin in that location, black meaning the port is empty, or red meaning that their is a port jam in that location
  • select the crystal you wish to mount by clicking on the circle associated with its cassette location (you will need to know this in advance of course!). It does not matter what the status of the location is. It can be either a green (known) or grey (unknown). In the topmost box in this interface, it should now show the cassette position and the crystal location next to the mount button as shown below left.

  • check that the indicator to the left of the mount button shows green (normal - above left). If it shows yellow (busy - above center) check just below this to see what the robot is doing. You will have to wait until the robot returns to the green (normal) state. If it shows red (reset - above right) then you will have to call User Support. If everything is ready, you can mount your crystal.
  • click the mount button and wait until the robot has gone through the mounting procedure. There may be times when it appears that nothing is happening but you should always see the yellow (busy) indicator and usually a short statement under this indicating what the robot is doing. You should see your crystal arrive on the screen to the right. Once it is there you can proceed to auto centering (see below) and collect your data.
  • there are two ways to dismount the crystal but you should always first check again to make sure the robot indicator is green (normal).

  • (i) click the dismount button to ask the robot to take the crystal and put it back in the cassette. The robot will return to the green (normal) state once this is done.
    (ii) if you have another crystal in the cassette that you wish to collect, simply select this crystal by clicking the location in the table. Then click mount and the robot will automatically dismount the crystal currently on the goniometer and mount your new sample.

Washing ice off the crystal with the robot

The crystal washing feature can be used to try to remove excessive ice from the crystal once it is mounted and before you start data collection. The button for crystal washing (see below left) is the third button in the Sample Automatic Mounting interface and appears just under the indicators showing the crystals selected for mounting and dismounting. During sample washing, the robot will dismount the crystal and place it back on the dumbbell magnet in the dewar. It will then pick up the magnet and move the sample in a circular motion and then up and down, before remounting it on the goniometer. You choose how many time you want the crystal washed by using the times pull-down menu (this does not mean the robot mounts and dismounts this many times, it is simply how many times it goes through the circular/up-down motion in the dewar). To start the washing process, press the Start Sample Washing button. The button will initially turn red (as shown below right) and you will see the warning Confirm may destroy sample. To activate washing, you need to click this button a second time within 2 seconds, otherwise the button reverts back to the grey Start Sample Washing button. This is to prevent accidentally initiating the sample washing.

You should be aware that it is possible to wash your crystal out of the loop, particularly if you are using large loops with small crystals.

Use this feature at your own risk!

Sample Annealing

It is known that protein crystals have an underlying granular (or mosaic) structure, giving rise to blocks of well-ordered crystalline lattice which produce sharp diffraction peaks, surrounded by disrupted water-rich regions which produce either diffuse scatter or no diffraction at all. The size of these blocks becomes smaller when the crystal is put under stress associated with flash-freezing (predominantly due to the expansion of the bulk solvent in the solvent channels) and this increase in microheterogeneity of the mosaic blocks leads to an increase in crystal mosaicity and a reduction in resolution and diffraction intensities. Crystal annealing, where the temperature of the frozen crystal is either raised to just above the freezing point of the cryoprotectant, or by several degrees but without thawing, is a method which have been used successfully to improve the diffraction quality of some flash-cooled crystals.

The most popular method of crystal annealing involves simply blocking the cryostream temporarily with a plastic card (the so-called "credit card" method). We have incorporated two methods for crystal annealing into Blu-ice, Flow Control annealing and Block Stream annealing. Both methods can be controlled remotely without the need to enter the hutch.

Flow Control Annealing

In Flow Control annealing, the cold stream can be temporarily stopped for a certain length of time specified by you. Only the cold stream is turned off, the outer dry nitrogen shroud remains on to protect the crystal from icing. To use this type of annealing, make sure the Flow Control tab is selected (this is the default upon first opening Blu-ice), select the annealing time using the anneal time pull-down menu as shown above, then choose the length of time you wish the cold stream to be off (the maximum time is 10 seconds but typically a time of between 2-5 seconds is all that is required). To start the annealing process, press the Start Sample Annealing button. The button will initially turn red (as shown below) and you will see the warning Confirm may destroy sample. To activate annealing, you need to click this button a second time within 2 seconds, otherwise the button reverts back to the grey Start Sample Annealing button. This is to prevent accidentally annealing the crystal.

Use this feature at your own risk!

Block Stream ("credit card") Annealing

This option is currently disabled due to incompatibility with other instrumentation.

Crystal Centering

  1. Select the Sample tab on the video display. (Or Sample profile on BL12-2).
  2. Select the zoom level Low to find the sample.
  3. Click on the sample to center it.
  4. Rotate phi 90 degrees by pressing the +90 or -90 button.
  5. Click on the sample once again to center.
  6. Select zoom level high.
  7. Repeat steps 2 to 4 to align the crystal accurately.
  8. If the crystal is difficult to see in some orientations, verify that is stays a center at different values of phi. It is possible to select different value by which to rotate the crystal from the drp down menu Rotate Phi

  • If you want to hit the crystal in a very precise spot and you cannot click on it accurately enough with the mouse you can use the "Move Sample" buttons. The double arrow buttons translate the sample horizontally by half a screen. The single arrow buttons translate the sample vertically or horizontally by the distance in microns displayed in the center of the buttons; you can select a different distance from the Move Sample drop down menu just above the buttons.
  • On BL12-2, a high magnification on-axis camera is available to align very small crystals. After roughly aligning the sample mount using the Sample Profile view, use this camera by selecting the Sample on Axis tab in the video widget. Make sure that the back light screen (see Sample Lighting below) is inserted for a proper sample illumination!

Auto Centering

  • The sample can be quickly aligned by selecting Center Loop.
  • This automated procedure sometimes fails if the lighting or sample shape is abnormal.
  • If the sample is asymmetric in the loop, a manual adjustment at zoom level High may be required.
  • Note: After auto centering is finished, the sample camera is not at the highest possible zoom level.

Adjusting Beam Size

  • To adjust the beam size click on the windows labeled Width and Height under the beam size label (see above). The white box on the Hutch monitor gets updated automatically to the input value. Like all other Blu-ice parameters, the color of this box becomes red if the current value is not same as the value in the input box.
  • With the exception of BL12-2, it is advisable to optimize the beam after changing the beam size to a smaller size. Use the Optimize Beam button located under the energy display to do this.
  • Clicking on the units will open a drop down menu that allows the units to be changed.
  • The beam size displayed is approximately the Full Width Half Maximum (FWHM) of the beam. Ideally the beam size should be the same or somewhat larger than the crystal size.
  • On BL12-2 the beam can be further collimated to a size of a few microns, using the microcollimator check button.

Adjusting Sample Lighting

To address the needs of remote users, an improved sample visualization system has been developed that provides remote control of the light intensity and direction. With this new system, during automated loop centering the lighting is automatically changed to the optimal settings for this process. Once loop centering is complete, the lighting is returned to configurable default settings. These settings may be adjusted using controls displayed in the Blu-ice GUI below.

    back lighting (profile camera)

    side lighting

    both lights on

This sample illumination system consists of two light sources:

  • Back lighting: a uniform back light, developed in house and composed of an array of 81 ultra-bright LEDs. This light typically shows as a blue background. With this lighting only, the loop is normally seen as a dark silhouette, as shown above in the first image. This is optimal for automated loop centering using the Center Loop button, or with automated loop centering used in conjunction with automated sample screening (see the Screening tab for further instructions). This light can either be switched on or off using the Back Light button. On BL12-2, a screen can be inserted behind the sample to provide back light illumination for the On-Axis camera. This screen is automatically removed during data collection.
  • Side lighting: a variable intensity side light comprised of an optical fiber bundle connected to a Schott Fostec model DCRIII halogen source. This source is directed at the crystal from an angle of 45 degrees, mounted above the X-ray beam. With this lighting, the background is generally dark and the loop and crystal are seen clearly, as shown in the second image above. The intensity of this lighting can be adjusted using the Side Light Intensity slidebar as shown below.
  • The two lights can be used in conjunction, and the intensity of the side light adjusted accordingly to provide optimum visualization of the crystal, as shown in the third image above.