The Sample Tab (Fig. 1) 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.
Figure 1. The Sample Tab. (Click-on to enlarge)
General directions for mounting a crystal in a cryoloop and flash cooling can be found in the Cryo-Tools Instruction Manual
- Move the detector and beamstop (Fig. 2) 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.
Warning: Do not attempt to adjust the beam stop manually.
Figure 2. Beamstop Control. Select largest distance for mounting samples manually.
- 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 ~10 mm and it should be centered).
- Mount your sample on the goniometer head.
- Use the resolution predictor to calculate the optimal sample to detector and 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 (Fig 3) may be used to mount crystals one at a time using the Stanford Automated Mounting (SAM) robot. While it is more efficient to automatically mount and screen samples with Sample Screening, use of the sample tab allows remounting samples for data collection without erasing previous screening results.
Warning: The Sample Tab sample mounting is not connected with the sample spreadsheet. The screening results from samples mounted from the sample tab will not be saved.
Figure 3. Sample Mounting Robot Control. The green ports have a sample, D7 and D8 (black) are empty. E1 (red) is jammed and cannot be mounted. C1 (magenta) is currently mounted. C3 (zigzag icon) will be mounted next.
The procedure for mounting and dismounting a crystal is as follows:
- Precool the tongs using the top right button in the Sample Mounting Robot Control widget. This step is not strictly necessary, since the robot will always cool the tongs if they are warm before fetching a sample, but if you do this while you are getting ready for the experiment, it will save you some time. If the robot status to the right of the Mount button is "Standby", the tongs are already cooled inside the sample storage dewar.
- Choose the correct position of your cassette (left, right or middle) by selecting the correct tab above the table. Usually, the locations of the sample pins will be shown in green, and the empty ports will be shown in black. A deep red port indicates that it is jammed, and the robot cannot retrieve the sample. Unprobed crystal locations in the table will be shown as gray circles. This means that the robot does not know whether a pin exists in that location or not. While this will not prevent you to mount the sample, some robot error conditions cause a loss in the probing information. Ask support staff if you notice a gray port in a location where you have samples.
- Select the crystal you wish to mount by clicking on the port associated with its cassette location. The topmost box in the interface, should now show display the cassette position and the crystal location to the left of the Mount button as shown below left. Also, a yellow zigzag line will appear over the selected sample.
- Check that the robot status indicator to the night of the Mount button shows green. If it shows "Busy" in yellow, the robot is doing something (returning a sample, drying the tongs, etc) and you will have to wait until the robot returns to the ready state. If the status is displayed in red, it means that there is an error. You will have to call User Support.
- Click Mount and wait until the robot has gone through the mounting procedure. The port will become light green when the sample is in the dumbbell magnet, and dark green when it is in transit to the goniometer. Once the sample is mounted, the port will turn magenta. At this point, ou can proceed to auto centering (see below) and collect your data.
- If you want to speed up mounting of the next sample you can "pre-fetch" it while the current sample is mounted. To do this, simply click on the next sample you wish to mount. You will see the yellow zigzag icon on that sample. If the robot is idle in standby, it will pull out the sample from the port and place it on the magnet, until you click Mount. Note that manual pre-fetching is not necessary in the screening tab. There, the next sample in the selection list is always pre-fetched automatically.
- Each sample will be automatically dismounted every time you mount a new sample. To dismount the last sample, use the Dismount button.
Warning: Use this feature at your own risk.
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 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 inside the dewar. It will then pick up the magnet and move the sample in a circular motion and then up and down within the liquid nitrogen bath, before remounting it on the goniometer.
You choose how many time you want the crystal washed by using the times pull-down menu (Fig. 4) 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 (Fig. 5) and you will see the warning Confirm May Lose Sample. To activate washing, you need to click this button a second time within 2 seconds, otherwise the button reverts back to the gray Start Sample Washing button. This is to prevent accidentally initiating the sample washing.
Figure 4. Specify the number of cycles within the liquid nitrogen tank
Figure 5. Confirmation to start annealing sample is required.
Warning: Be aware that it is possible to wash your crystal out of the loop, particularly if you are using large loops with small crystals.
For information on how to prepare and ship un-frozen samples, please see the instruction manual on Remote Access at Elevated Temperatures and Controlled Humidity.
A crystal dehydration experiment may improve the diffraction resolution of ill-behaved macromolecular crystals. During these experiments, crystals are equilibrated at decreasing values of relative humidity (RH) in a stepwise fashion. Diffraction images are collected at each step to compare the diffraction quality and resolution.
The Arinax HC-lab is a scientific instrument designed to regulate the relative humidity of macromolecules crystals while mounted on the goniometer. Installed in the same way as a standard cryogenic stream head, the HC-lab blows a controlled humid air stream onto the sample. The air stream is generated by using the Dew Point Method to remove water from a hot and saturated air stream so that it will be at the correct RH when it reaches the crystal. The device provides a user-friendly sample environment to collect data at room temperature without the need of capillary mounting. It also enables crystal dehydration experiments with the aim of improving crystal diffraction.
The HC-lab comprises the machine body connected via a 1.5 m tube to a nozzle, which points the humid stream onto the sample (Figure 1).
Figure 1. Setting of the HC-lab at the SSRL beamlines.
Any RH value between 99.8% and 50.0% can be set using the Humidity control in the Blu-ice Sample Tab interface (Figure 2). If you cannot see the humidity control options, ask your user-support scientist to enable humidity control for your experiment. Once the RH value is set, wait until the air stream reaches this value and it is stable
Figure 2. Humidity Control Tab within the Blu-Ice Sample Tab.
The green area displays the current relative humidity. To change the set point, type a value in the entry box (which will be in red text) then click “Move” to change in set point (the “set point” text will then turn black).
Further integration of the HC-lab control software into the Blu-Ice software is in progress.
Regardless the type of experiment you are planning, the first step is to measure or estimate the RH value of your crystallization solution.
Measuring the RH Starting Point
- Center an empty loop perpendicular to the plane of the image and put a drop of the mother liquor (crystallization condition) on it.
- Observe the drop size behavior: if the drop size increases/decreases the mother liquor RH is below/above the humid flow RH value, respectively.
- Adjust the RH set point until the drop size remains stable in time. This can take several minutes. Once the drop size is stable, the mother liquor and the humid flow have the same RH value. This RH value is the starting point for RT data collection or dehydration experiments.
Estimating the RH Starting Point
- An estimate of the the RH Starting Point value can be based on the composition of the crystallization condition. The RH values for the most common precipitants and cryo-protectants have been empirically measured in this Article.
- For a more accurate estimate, ESRF has developed a Series of Equations that can be used to determine RH Starting Point values for your experiment.
- Set the RH value to the appropriate starting point.
- Mount and center a crystal.
- It is best if any excess of mother liquor around the crystal is removed with a paper wick.
- Collect a diffraction image to characterize the initial state (space group, unit cell, mosaicity, etc).
- Decrease the RH value by steps of 1%. At each RH step let the crystal reach the equilibrium. It takes about 5 min to stabilize the system (reaching the set point, stabilizing the drop size).
- Collect a diffraction image in order to compare it with the previous ones.
- In case of improved diffraction quality further optimization may be necessary, e.g. chose smaller step sizes for the RH set points.
Complete datasets may be collected from crystals at optimized humidity. Multiple crystals may be required.
Alternatively, a new rapid nozzle switcher has been recently developed at SSRL that will be installed on some of the crystallography beamlines. Using this device, crystals at optimized humidity can be flash-frozen to collect complete datasets.
Warning: Use this feature at your own risk.
It is known that protein crystals have an underlying granular (or mosaic) structure, giving rise to blocks of well-ordered crystaline 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 micro-heterogeneity 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 cryo-protectant, 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.
Currently, only one automated method for crystal annealing is implemented in Blu-Ice: Flow Control (Fig 6).
Figure 6. Cold Stream flow control annealing of sample.
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 (Fig. 7). To activate annealing, you need to click this button a second time within 2 seconds, otherwise the button reverts back to the gray Start Sample Annealing button. This is to prevent accidentally annealing the crystal.
Figure 7. Confirmation to start annealing sample is required.
Select the Sample Tab on the video display (or Sample Profile on BL12-2) to access the Video Control Panel (Fig. 8).
Figure 8. Video Control Panel.
To center a crystal:
- Select the Zoom Level Low to find the sample.
- Click on the sample to center it.
- Rotate phi 90 degrees by pressing the +90 or -90 button.
- Click on the sample once again to center.
- Select Zoom Level High.
- Repeat steps 2 to 4 to align the crystal accurately.
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 Rotate Phi drop down menu.
If you want to move the crystal in a very precise location 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 view along the X-ray path is available to align very small crystals. After roughly aligning the sample using the on-axis view by selecting the Sample on Axis tab in the Video widget.
Important: Make sure that the back light screen is inserted for a proper sample illumination (see Sample Lighting below).
The sample can be quickly aligned by selecting the Center Loop button .
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 (Fig. 9).
Figure 9. After loop centering, the magnification is set to view the loop.
To adjust the beam size, click on the windows labeled Width and Height under the Beam Size label (Fig. 10).
Figure 10. Beam size control.
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 Microbeam Collimator check button.
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 Blu-Ice.
This sample illumination system consists of two light sources that can be used independently or together:
- Back lighting (Fig. 11): 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 (Fig. 12). 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.
Figure 11. Back lighting.
Figure 12. Back and side light control.
- 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 (Fig. 13). The intensity of this lighting can be adjusted using the Side Light Intensity slidebar (Fig. 11).
Figure 13. Side lighting.
- The two lights can be used in conjunction (Fig. 14) and the intensity of the side light adjusted accordingly to provide optimum visualization of the crystal.
Figure 14. Back and side lighting on.