These pages describe how to use the Stanford Auto-Mounter (SAM) System, SAM sample pins, SAM cassettes, Uni-Pucks and associated tools.  For information on the SAM hardware, including design drawings and vendors for the SAM Cassette Kit, see the SAM Development pages.

SAM is a completely integrated hardware and software system for mounting and dismounting pre-frozen protein crystals and screening samples for x-ray diffraction quality in a fully automated or semi-automated fashion. SAM is installed on all of the SSRL macromolecular crystallography beam lines and is seamlessly integrated into the beamline control and data analysis software. Since upgrading the robot system in 2014, the screening sequence (comprising crystal mounting, automatic sample loop centering in the x-ray beam, video and diffraction image acquisition at 0 and 90 degrees, and dismounting) may take less than one minute per crystal. The diffraction images are analyzed and autoindexed on the fly.

Samples may be stored in either SSRL cassettes or Uni-Pucks (Fig. 1) for use with SAM. If you are scheduled beam time with use of the robot sample mounting system, a cassette kit can be lent to you. A kit can be requested by filling out a SAM Cassette Kit Request.

The following sections describe how to prepare your crystals for data collection using the SSRL robotic sample mounting system. Follow these instructions to prepare the sample pins, mount them in an SSRL cassette or Uni-Puck and ship them to the SSRL.

For additional information on the SAM system, see also Automated Sample Screening in Experimental Goals. There are also Video Tutorials illustrating Sample Preparation; sample container loading using SSRL Cassettes and Unipucks; and use of the SAM system for data collection.

For information about the cassette kit tools, including drawings and vendor information, see the see the SAM Development pages.

To have a successful experiment proper sample pin preparation is essential. The majority of problems we have observed with SAM have been related to use of improper sample pins. To avoid these problems please read the following directions.

The SSRL system supports only Hampton-style CrystalCap Copper Magnetic pins or CrystalCap Magnetic pins. The allowed pins sizes are 16 or 18 mm (Fig. 2). The 18 mm size copper pin is preferred.

Warning: Do not exceed the maximum microtube length shown in the diagram. Unacceptable lengths are marked with a red x. The 11 mm MiTeGen MicroMounts and meshes should be used with the CrystalCap Copper pin base; longer MicroMounts will need to be cut to the proper length before inserting into this pin base.

Compatible Hampton-style sample pins (Fig 3A) may be purchased from a number of vendors including Hampton Research, MiTeGen Crystal Positioning Systems, or Molecular Dimensions.

If sample pins are purchased from MiTeGen, use the B1, B1A or B3S with the 18 mm MicroMounts, MicroLoops, MicroMeshes or MicroGrippers (Fig 3B). The B1, B1A, B3A and B3 bases can also be used with 19 mm mounts. The older B2 base can be used with 11 mm MicroMounts and 10 mm nylon loop mounts.
 
Note: The SSRL loop-centering routine, based on visual analysis, is not yet optimized for use with the MiTeGen MicroMount, these mounts, as well as the loops are well suited for Rastering. Molecular Dimension ActiLoops and LithoLoops are also compatible with the robot, if mounted on a solid (non CryoCap) base.

Warning: SPINE-standard pins can not be used with SAM.

Note: We recommend using Hampton-style Copper Magnetic pins because no laser etched lines are exposed. These are the pins supplied with the cassette kit. The MicroTubes used with Magnetic pins can sometimes break off if bumped (Fig. 4).

MicroTubes should be affixed inside sample pins using epoxy. Any epoxy with a curing time between 5 minutes and 24 hours should work well for this purpose. We have found that superglue and superglue gel is less reliable than epoxy for affixing MicroTubes. A number of failures with the SAM system have been attributed to using other types of adhesives on sample pins such as wax, nail polish, and Duco cement and these should not be used (Fig. 5).

Warning: If an adhesive other than Epoxy has been used to affix the MicroTubes to your sample pins, please let your user-support person know in advance of using them. These pins may cause problems with the SAM system.

Be careful not to use pins with excess epoxy on the pin base, post and pin and do not get grease or excess cryo-protectant on the pin body (Fig. 6). At liquid nitrogen temperatures, grease from crystallization trays gets rock hard. If you continually reuse your pins, please also inspect them for corrosion and loose MicroTubes. Anything that changes the outside form factor of the sample pin could cause the pin not to fit properly in the SAM robot tongs.

If you would like to mark your sample pins different colors, use permanent marker for this purpose. Paint or nail polish should not be used as this can change the form-factor of the pin or be sticky. Hampton Research now sells a new pre-assembled Copper Magnetic sample pin (catalog number HR5-112) which is already color coded according to the size of the nylon loop attached. It also has an alpha numeric code and bar code. See Bar-Coded Pin to learn how it can be used as a cassette or Uni-puck identifier ID.

All pins must be tested to ensure they fall within the allowed tolerances. We have found some irregular pins that if used would damage the SSRL sample mounting system. To test your pins, place them on the end of the Pin Tester magnetic tool on the red line (Fig. 7). Only use pins that completely cover the red mark without forcing.

Warning: Pins that fail this test should not be used!

Broken micro-tubes (Fig. 4) and torn nylon loops are usually a result of mishandling sample pins in preparation for reuse. In particular pins should not be piled together in a container when washing and drying them. To wash pins, place them individually on a magnetic tray. Hang the tray upside down to dry the pins. It is important to store your sample pins in a safe place when they are not in use. The microtube storage rack (Fig.8), available through Fisher, is a useful storage location for extra pins.

A bar-coded sample pin base may be used for sample container identification (Fig. 9).

These pin bases can be supplied to you by MC staff. When using a cassette, the bar-coded pin base should be placed in the A1 port (Fig. 10).  When using a uni-puck, the bar-coded pin base should be placed in the first port, labeled with the number 1 (Fig. 10). Bar-coded pins in other locations will not be used for identification. Do not glue the bar-coded pin inside the container as this will prevent the SAM robot from removing the pin to scan it.

For container identification, the SAM robot removes the bar-coded pin and reads it using a bar-code scanner installed at the beamline (Fig. 11).

If samples are mounted on the bar-coded pin base, they can be mounted for screening and data collection.  Reading the bar-code does not jeopardize the safety of the sample.  However, we recommend not using a sample on the bar-coded pin base because this will help prevent the bar-coded pin from being lost or misplaced during the sample preparation process.

If you are a new user scheduled to use the SAM robotic system, you can Request a Cassette Kit.  For additional information about the kit tools, including drawings and vendor information, read about the SAM Development.

The cassette contains 96 sample ports each port contains a ring magnet which holds in the sample pin. A cutaway view of the cassette is shown in Fig. 13. The ring magnets are shown in green. The magnets are held in place by a polycarbonate washer shown in white. The washers are removable so any broken ring magnets may be replaced.

Cassettes should not be stored where magnetic debris can get inside the ports. Before using your cassette, it should be inspected to ensure the ports are all empty.

The transfer handle is used to safely transport cold cassettes. To attach the transfer handle (D) to a sample cassette (A) first place the locking pins into the slots at the top of the cassette (Fig. 14). Then push down the handle, and rotate clockwise until the handle locks in place.

The slotted guide tool (Fig 15) consists of a long magnetic wand with two slots in the middle and a loading guide with a track on the handle that accommodates the slots on the wand tool. To flash freeze samples, the wand may be inserted into the top of the loading guide. To transfer pre-frozen samples it is important to keep the samples at liquid nitrogen temperatures during transport into the cassette. The wand should be inserted from the side of the slotted guide tool to keep the sample under liquid nitrogen during transfer. Use the slot closest to the red (or maroon) side of wand (weak magnet) for loading samples.

When unloading samples the wand is turned around and the opposite slot and stronger magnet is used.

The circular cutout in the loading dewar may be used to hold samples in cryo-vials or pucks for transfer.

The most common problems we see observe with cassette loading is the accumulation of ice in the liquid nitrogen bath. Ice will stick to your sample as it is transferred through the liquid nitrogen on the way to the cassette port. It can also fall inside the cassette ports. If excess ice is observed in the bath, the cassette should be stored in a cold dry-shipping dewar and the loading dewar emptied, dried and refilled with liquid nitrogen before proceeding.

Warning: To prevent ice from accumulating in the liquid nitrogen, the cassette should not be stored in the loading dewar for more than 20 minutes without exchanging the liquid nitrogen.

To avoid ice accumulation, it may be useful to to load cassettes under a fume hood or a dry box (as an example, see a description of Nham Nguyen's Cassette Loading Setup. If not using a fume hood or a dry box, cover the dewar with the lid when you are not mounting crystals. To prevent ice from falling into empty cassette ports, some users advise filling empty ports with blank pins. Each blank pin is removed just before inserting a sample pin into the port.

Safety: Please make sure you are familiar with the liquid nitrogen safety procedures at your institution before carrying out these procedures.

1. The custom foam dewar should be filled up to the internal indicator ledge (Fig 16). This takes about 4 Liters of liquid nitrogen; it will be necessary to top off the liquid nitrogen level after inserting a warm cassette.

2.   Once the dewar is filled and equilibrated, place the cassette in the dewar and tilt the transfer handle until it rests inside the side notch. Then push the cassette forward (Fig 17a) until the bottom of the cassette is touching the edge of the dewar (Fig. 17b).  

3. The Guide Tool may be pre-cooled on the side dewar shelf (Fig. 17b). To prevent condensation on the metal portions of the guide, it may be stored on the shelf between loading samples.

4. Rotate the sample cassette with the transfer handle to access the desired cassette port.

5. Place the guide tool (F) on the cassette centered on the port (Fig. 18A).

6. To flash freeze a sample directly into a Cassette:

1. Put a Sample Pin onto the red side of the Magnet Tool (E) (Fig. 18B) and pick up a crystal (Fig. 18C).

2. Flash-freeze the crystal by placing the pin through the handle of guide tool and into the cassette port, minimizing the time the crystal is in the air (Fig. 19).

7.  To transfer a pre-frozen sample from a vial (or puck) to a cassette:

1. Under liquid nitrogen,tilt the Vial and use the red side of the Magnetic Wand tool to remove the Sample Pin (Fig. 20B). Be careful to keep the sample under liquid nitrogen at all times (Vals or Uni-Pucks may be placed in the round cutout inside the dewar).

2. Slide the lower slot of the Magnetic Wand tool into the guide tool (Fig. 21) while keeping the sample under liquid nitrogen.

3. Once the Magnetic Wand Tool is in the center of the Guide Tool, the wand tool may be pressed against the back of the guide and pushed downward placing the pin into the port. (It is sometimes helpful to rotate the wand while it is pushed down to break any ice that may have formed between the Magnetic Wand Tool and Sample Pin.) The Magnetic Wand Tool may then be pulled up, removed and the next sample transferred. 

Advisory: As you fill the cassette, it is highly recommend that you record the location of each crystal as described in Storing Sample Information in the Sample Database.

Note: A separate file should be created for each cassette.

Advisory: It is strongly recommended to Test the Shipping Dewar prior to shipping samples to make sure that the samples will be kept cold before arrival. 

1. Prepare the Dewar for Shipping. Place the Teflon Support Ring (C) inside the shipping dewar (Fig. 22) before inserting the Canister (G) and fill the dewar with liquid nitrogen in the usual manner.

2.  Transfer the cassette into the canister in the dewar (Fig 23), minimizing the time the cassette is in the air. Remove the transfer handle from the cassette by pushing down on handle and turning counter-clockwise to     release it.

3. Two cassettes may be shipped inside one canister. When shipping just one cassette, place the Styrofoam spacer (F) on top of the cassette to keep the cassette in place during shipment (Fig. 24).  

  The cassettes are compatible with most dry shipping dewars.

Advisory: We recommend using the combination of a MVE model SC4/2V, Taylor Wharton CX100 or CXR100R cryogenic shipping dewar with a Taylor Wharton TAY CX10-8C00 dewar container.

For information on how to ship dewars to and from SSRL, see Shipping Dewars.

For information on how to use the Uni-Puck, see Loading and Shipping Samples in the Uni-Puck.

Advisory: It is strongly recommended to Test the Shipping Dewar prior to shipping samples to make sure that the samples will be kept cold before arrival.

Sample information can be entered into an Excel spreadsheet and then uploaded and stored in the Sample Database. This information is used by the data collection software and all screening results are written back to the database. Information in the Sample Database can be viewed from the Sample Database Interface or in the Blu-Ice data collection software.  The information can also be downloaded back into an excel file using the web interface.

An option to use a Default Spreadsheet to store information about your experiment without downloading a template file is also available via the Sample Database Interface.  To learn more about this option, skip to Using a Default Spreadsheet.

The following instructions explain how to download and fill in a sample spreadsheet:

1. Use the following links to download a blank spreadsheet (see Fig. 25 for an example template). A different spreadsheet format is required for each type of container used to send samples to SSRL.  For samples at cryogenic temperature, use the SSRL Cassette or Uni-puck template (four pucks per spreadsheet) or for samples at controlled humidity, use the SSRL Plate template. These links can also be found in the Sample Database Interface.

• SSRL Cassette Excel Template
• Uni-Puck Excel  Template
• SSRL Plate Excel Template

If your browser cannot display Excel spreadsheets, click the Save to File button in the pop-up menu; navigate in the dialog box to the directory of choice and click Save.

If you are shipping more than one cassette or plate, use one spreadsheet per cassette or plate.

A single Uni-puck spreadsheet holds information for up to four (or less) pucks (labeled A, B, C and D).

2.  Type in information for samples. Fig. 26 shows a partially filled out spreadsheet.

Important:  Verify that "text" formatting is being used.

Critical: Do not add or remove rows from the spreadsheet, do not edit the Port column or heading names in any manner.

Note: A new heading and column can be added to the Excel spreadsheet however, these columns will not be uploaded to the Sample Database.

Descriptions of the spreadsheet columns:

1. ContainerID: Uniquely identifies the cassette, puck or plate. This information is useful for staff to verify the correct spreadsheet is assigned to the correct container on the beamline.

Recommendation: Use the ID number that is already engraved on your cassette or puck.

2. ContainerType: The type of sample container whch is prefilled in the spreadsheet templates.
3. Port: Indicates the position of the sample in the sample container. Do not edit this column in any manner.  If the Sorting Tab is used in Blu-Ice to move samples from occupied ports to unoccupied ports, the moves will be recorded in a new column called Move.
4. Barcode: Bar-Coded Pins can be used for access protection or for recording physical IDs of individual samples.  Although the A1 pin can be used for samples, it is also reserved for identifying the container and assigning an account owner to the container.  For cassettes and plates, the barcoded pin should be placed in the A1 or 1 position, respectively. For pucks, the barcoded pin can be placed in any one of the A1 positions.  When support staff first loads the cassette for a user, the A1 port is automatically scanned and staff assigns the barcode to your account. Future scans of this barcode will assign your account automatically. The system can also scan all sample pins for barcodes, however, this feature is not available by default.  Please contact your assigned support staff if you want all your pins to be scanned during screening.
5. CrystalID: Used by the Screening System to create root filenames. By default, this column is prefilled with the Port name.
6. Directory: This column specifies the root directory name. By default, this column is prefilled with the Port name.

Warning: Only use alphanumeric characters with no blank spaces for the CrystalID and Directory. Do not use special characters, brackets or spaces since they are not allowed to be used in Linux file or directory names.

7. The remaining columns are intended to help with sample tracking and storing sample parameters. Filling in these columns is optional. The allowed headings are: Protein, Comment, CrystalCond, FreezingCond, Metal, and Priority.
8. Once Screening is run on samples, additional columns and information will be recorded in the Sample Database which can be downloaded into an Excel spreadsheet.  The headings are: Score, UnitCell, Mosaicity, Rmsr, Resolution, Isigma, BravaisLattice, Solution, SystemWarning, AutoindexDir, and AutoindexImages.

3.  After downloading or editing the spreadsheet, verify that it is saved as a 'Microsoft Excel Worksheet' with an .xlsx extension.

Once the Excel spreadsheet has been filled out it can be uploaded to the Sample Database via the Sample Database Interface:  If your beam time has started and you have been enabled to start the experiment, it is also possible to launch the Sample Database Interface from Blu-Ice: Click on "Spreadsheet" under the Web button in the Screening tab of Blu-Ice.

1. Log into the Sample Database Interface using your MC account name and password.
2. Once you are logged into the system, select the New Spreadsheets tab (Fig 27).

3. Under Step 2), click on the Upload a spreadsheet from your computer link.
4. Click on the Browse,,, button to find the Excel template file. If you have trouble locating the file, verify that the browse filter is not filtering out the file. Once the file is selected in the dialog box, click Open.
5. Enter a name for the Excel sheet or use the default name (Sheet1). Click Upload and the uploaded data will now be displayed (Fig. 28). If your spreadsheet contains a formatting error, the file may not upload.  Because some formatting errors will cause only part of the spreadsheet to be uploaded, verify your spreadsheet information is intact.

Spreadsheets can be removed from the Sample Database by clicking on the SpreadsheetID and then the Delete button at the bottom of the "About..." page in the interface (Fig, 29).

Under the My Spreadsheets tab in the Sample Database Interface, click on the link for a particular spreadsheet. Next, click on the Samples link under the "View Samples" heading. Only columns with data will be displayed.

Sample screening without uploading a spreadsheet is possible. On the New Spreadsheets tab in the Sample Database Interface, select the appropriate link under "Default Spreadsheets".  A new spreadsheet will be created directly in the Sample Database.

The information in the Sample Database may be assigned to a beamline once beamline access has been permitted. On the My Uploaded Spreadsheets tab, click on the Assign button for the spreadsheet that you want to assign to a beamline. A total of 3 cassettes can be placed in the experimental hutch storage dewar and they are labeled Left, Middle and Right. Select the appropriate beamline and position (support staff will let you know which position to use) using the drop down menu (Fig, 30). If the assignment fails, ask support staff to verify that your account has permission to access the beamline.

The spreadsheet assignment can also be carried out in the Screening tab of Blu-Ice. See Uploading Sample Information for more information.

To make changes to a spreadsheet that has already been uploaded to the Sample Database, first, download the spreadsheet to an Excel file on a local computer, then edit it in Excel  To do this, click on the My Spreadsheet Uploads tab.  Click on the link for the spreadsheet that requires editing.  An About... page will appear providing information about the spreadsheet (Fig. 29).  Under the Downloads heading, click on the Download Spreadsheet link.  The file can be edited in the browser if it supports excel type documents, or it can be downloaded to a local computer.

You can edit the Excel file on the Unix beamline computer with the LibreOffice software. To run LibreOffice from the beamline computers type:

After finishing editing the spreadsheet, upload the modified file to the database as described in the Uploading an Excel Spreadsheet to the Sample Database section above.

Alternatively, during the experiment, Blu-Ice and Web-Ice can be used to directly edit selected fields..To learn more about editing spreadsheet information using these applications, consult the Blu-Ice and Web-Ice Manuals, respectively.