Skyscan 1172 Bone Users Manual Indiana University School of Medicine Updated August 7, 2009 Although this is meant to be a stand-alone document which will allow users the essential information to perform, reconstruct, and analyze scans of skeletal tissue, it is by no means as comprehensive as the manuals provided with the scanner. Those are located on the scanner computer C drive (Skyscan/Manuals). Turn on the system If the system is not already on, turn the scanner on by turning the key, located on the right side of the machine, to start (it will automatically rebound to ‘on’). Once the scanner is on, double click on the ‘Skyscan-1172’ icon located on the computer desktop. Be sure to fill out the use log hanging on the wall above the scanner computer. Once the software opens, you will see the following window. Click on the ‘X-ray’ button (second icon from the left on the top toolbar). If the scanner has been off for >8 hours, the system will need to warm-up, which occurs automatically if necessary (a window will open telling you the status of the warm-up). This will take approximately 15 minutes and the x-ray source will automatically go off once it is complete. If the machine has been used within the last 8 hours, the x-ray will automatically come on and no additional pop-up window will appear). Specimen preparation We have three specimen stages, two of which are shown here (plus one larger). The smallest has a diameter of 20 mm, the medium one has a diameter of 40 mm, the larger (not shown) has a diameter of 60 mm. There are also ‘posts’ which can be used in combination with Styrofoam holders or other vials. When positioning the specimen, keep in mind that maximizing the vertical orientation is important, otherwise reconstruction contouring will be more work. Positioning a long bone (e.g. femur) distal end down will result in reconstructed images being x-sections through the bones. While this is probably the most preferable orientation, it makes it difficult/impossible to scan more than one specimen at a time. It is possible to scan the bones horizontal, and then to ‘reslice’ them in a different orientation later on (described in the reconstruction instructions). Multiple long bones can then be scanned at once using the batch scanning protocol (see section on Batch scanning). Small bones such as vertebra can be scanning using the batch scanning method in either horizontal or vertical orientation. Once your specimen is fixed to the stage, open the scanner door (the far left icon in the toolbar (door) and insert the peg into the holder (shown to the right). Tighten the holder by turning clockwise. Be careful not to push on the holder, this could result in poor alignment of the holder. DO NOT OVERTIGHTEN. Close the scanner door by clicking on the door icon. Setting up the scanning parameters (Voltage, resolution, region of interest) Turn the x-ray tube ‘on’ by clicking on the ‘X-ray’ button (second from the left, in the toolbar). Adjust the voltage for the scan by selecting ‘Option-X-ray source’ from the top menu. The following window will open: Use the gray slide bar under ‘voltage’ to pick the voltage (kV) you wish to use. Hit ‘apply’ and the blue bar will move to the level you have selected. You can then close this window (use the red x in the upper right corner). You will notice that in the digital display on the Skyscan machine, the kV will read approximately what you selected (a good double check that you are at the correct voltage). It is not uncommon for this readout on the machine to be slightly different than the voltage you told the machine to be at (for example if you set the system at 60kV, the digital display might read 61kV). The best thing is to always set the system at the voltage you want, and don’t worry too much about the readout on the digital display. Each time you open the door (e.g. to change specimens) the system resets the x-ray to a default. For unknown reasons, sometimes the default x-ray is 60, other times it is 70. So, you need to be judicious that every time to turn the x-ray on, make sure you adjust the x-ray intensity to the proper level. The best way is probably to leave the x-ray tube window open to remind you to watch. Resolution (µm) and speed (scan duration) are inversely related. If you want high resolution, the scan will take longer. It is important to consider what resolution is ‘necessary’ to obtain the data you want as lower resolutions are not always better. The resolution of the scan will be determined by the amount of ‘zoom’ and the camera ‘binning’ mode. The binning mode is displayed in the fourth section of the bottom toolbar (e.g. 1000x524 above). The 1000 (referred to as 1K) is the lowest setting, with the 2000 (2K) being medium and the 4000 (4K) being the highest. To understand how these relate to the resolution, change from 1K to 2K (click on the box that says 1000x524), and other options will appear. Resolution range for 1K is 6.4 to 35 um. Resolution range for 2K is 3.2 to 17 um Resolution range for 4K is 0.8 um to 8 um. As you can see, it is possible to scan at 8 µm using any of the binning modes. For a qualitative comparison, see the file ‘Mouse femur scanning’. The key to remember about the binning modes is that it is simply combining pixels. At the 4K mode, all camera pixels are used while at the 2K mode 2x2 pixels are combined and at the 1K mode 4x4 pixels are combined. Scanning time (and file size) is 4K > 2K > 1K. The determining your resolution, move the specimen into the field of view. To do this, use the continuous imaging mode (click on the TV screen icon, fourth from the left). To move the specimen into the field of view you will need to move the stage. This is done by using the green arrows in the tool bar at the bottom of the screen (see image below). To move slow, click on the arrows (next to the 0.000mm readout). To move faster, double click on the number and enter a position. Move the specimen up (most likely) to be in the field of view (0 is lowest position, 50 is maximum). Since you are in live image mode you will be able to see the specimen as it moves up/down. If you want to rotate the specimen, use can do so using the second slide bar (the one with the turning arrow). If you are in live image mode you can see it turn. This tool will give the degree of rotation for the sample. It is a good idea, once you have the resolution of your scan set, to rotate the image to assure that in all orientations the entire sample is within the field of view. Return the rotation of the stage to 0 degrees once you are done. Bone should always be scanned with the Aluminum 0.5 mm filter in. This cuts the low energy x-rays out of the scan and reduces beam hardening. To insert/remove a filter, there is a region next to the ‘start flag’ in the bottom toolbar. A check notes the current set-up. This parameter defaults to whatever was used by the last scan. Thus, it is always good to double check this for your first scan of the session (after that it will not change on its own). Flat field optimization Flat fields are used to ‘correct’ the images recorded by the camera by accounting for any deviations in the x-ray source or camera at a given setting on a given day. If you are running a series of scans that are all at the same setting, you need to run a flat field correction at the start of the scanning session, but NOT before each scan. However, if you are scanning a few samples at one setting, and then switch to another set of samples at another setting, you would have to run a new flat field when you switch. As the flat field correction is to account for deviations in the scanner on a given day, there is no reason to save these (you have the option later). That is, run a new flat field each day. The general procedure is: 1) Remove sample from field of view (write down the position it is in first!). To do this, double click on the up/down position arrow at the bottom and enter ‘0’. If the specimen is too large, you must remove it altogether (assure when you then turn the x-ray back on, you reset the voltage and all the other parameters to the proper level). 2) Turn off flat-field correction (Options-Preferences). Uncheck both marks next to the “Flat-Field correction” (see image to right) 3) Hit ‘Alt+Ctrl+Shift+S’. Nothing will happen…but you need to do this. 4) Hit ‘Alt+Ctrl+Shift+H’. The scanner setting window will open (see image to the right). If it doesn’t open, hit ‘Alt+Ctrl+Shift+S again, then ‘Alt+Ctrl+Shift+H’.. Below the words “Aquire flat field reference” there is a button that says “Bright + Dark”. Select this and the software will take ~ 1 minute to acquire a flat field. Once it is done, hit ‘OK’. DO NOT CHANGE ANY OTHER NUMBERS IN THIS WINDOW. 5) Open the Preferences (Options-Preferences) and turn flat field corrections back on by placing a check in the two boxes you unchecked in step 2. 6) Obtain a still image (click camera icon on top tool bar). If lines do not appear on the image than right click with your cursor on the image. The Av should be >90%. 7) Move your specimen back into position (double click on the up/down arrow at the bottom and enter the number you wrote down in step 1). 8) Grab still image. Right click on the image to make the lines appear. The min% value of the transmission line should be 20-40%. If not, then choose a different voltage (higher if image is too dark, lower if image is too light) and repeat steps 1-10. Starting the Scan Once you are set to go, hit the “turning arrow key” on the upper toolbar (sixth icon to from the left). The following screen will open: **While this window can be opened using Options-Acquisition, doing it this way will not actually allow you to start a scan. **Always use a “_” at the end of the filename. Choose a folder to save the scans. All scans should be in a folder named as the name of the person scanning and be in the results folder on the D drive. Rotation step is the number of degrees the specimen rotates for each image. Increasing this number make the scan faster, reducing it makes the scan slower. Keep checks next to ‘averaging’ and x-ray OFF after scanning. There is no reason to have any of the others checked unless extenuating circumstances dictate (such as needing camera offset to increase scan width). Be careful when running scans one after another that these settings will return to their default each time so you will have to change them to your settings. Your scan time will be shown at the bottom. Hit ‘ok’ once you are ready to go. Batch scanning In order to scan multiple bones, or to scan multiple fields in the vertical direction (e.g. to scan an entire rat long bone), an ‘Oversize’ scan must be done. The set-up procedure is identical to running a single scan. Thus, setting up the orientation, optimizing the position of the specimen, and running flat fields are all the same. Once you have all of the standard parameters set up, go to ‘Actions’ menu and select ‘Set Oversize scan’. A new window will open. Click the ‘start’ button on the top. The system will now acquire a ‘scout’ view of the entire field of view. Once all the samples can be observed, you can hit ‘stop’. Within the scout view window, you can scroll up and down using the arrows on the right side of the window. To scan a single specimen that is large in the vertical direction: 1) Type in the name of the sample in the ‘Prefix’. This should end with a “_”. In the example to the left, the name of the bone is ‘tibia_’. 2) Click the ‘Top’ button and then move the cursor over to the image window and click above the top of your specimen. 3) Click the ‘Bottom’ button and then move the cursor over to the image window and click below the bottom of your specimen. 4) You will see the number of ‘Segments’ (for a single bone it will always be 1) and then the number of ‘parts’ (in this example it is 5). A ‘segment’ is essentially the bone you are scanning. A ‘part’ is how many scans it will do to encompass the entire bone. 5) If you mess up at any point, click ‘delete’ and start all over. 6) Once you are set, click ‘Start Scan’ and the ‘Acquisition window’ will open. DO NOT CHANGE THE FILENAME IN THIS WINDOW. You can change the folder location, as well as any other parameters such as rotation step. 7) Hit OK to start the scan. To scan a multiple specimens: 1) Type in the name of the first sample in the ‘Prefix’. This should end with a “_”. In this example, the first bone is ‘LV_’. 2) Click the ‘Top’ button and then move the cursor over to the image window and click above the top of your specimen. 3) Click the ‘Bottom’ button and then move the cursor over to the image window and click below the bottom of your specimen. If you select a bottom location anywhere in the box that appears after you select the ‘Top’, this will be a single ‘part’ (e.g. 1 scan). Selecting just a little beyond will add an additional part, and therefore increase your scan time. 4) To select another bone (‘Tib_’ in this example), enter the title in the Prefix, and then repeat steps 2 and 3. This can be repeated as often as necessary to get scan all your specimens. 5) If you mess up at any point, click ‘delete’ and start all over. 6) Once you are set, click ‘Start Scan’ and the ‘Acquisition window’ will open. DO NOT CHANGE THE FILENAME IN THIS WINDOW. You can change the folder location, as well as any other parameters such as rotation step. 7) Hit OK to start the scan. Turning off the system Once you are finished scanning, close the ‘Skyscan-1172’ software. You will be asked if you want to save the flat fields, select ‘No’. Once the software is closed, turn the machine off using the key which you used to turn it on. Log your time on the log sheet next to the keyboard. Notes: Data should not be kept on the system longer than 1 week. Please keep up on transferring files to external drives as the potential exists to have the data deleted if the system becomes full.