Method Notes 1172

May 23, 2008 METHOD NOTES, SKYSCAN 1172 DESKTOP MICRO‐CT

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Method Notes, SkyScan 1172 icro-CT: scanner operation m

This “method notes” document for the SkyScan 1172 micro‐CT scanner is a resource supplementary to the 1172 scanner manual. It is intended to provide step‐by‐step uidance for the procedures associated with making scans and adjusting and gmaintaining the scanner for optimal performance. ollowing the procedures in this document will help you get the best 3D imaging esults from your micro‐CT scanner. Fr


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C

ontents

1. Before you scan 3 2. Turning the scanner on and off 4 3. Refresh the flat field correction for the current scanning mode 8 13 4. Doing a scan

5. The configuration files: a fast and easy way to change applied voltage 18

6. Adjusting the x‐ray camera exposure time for a scanning mode 23 7. Changing the applied voltage 29 8. Examples of improvements to projection image contrast and

quality, achieved by adjusting filter and applied voltage 33 9. Setting up to scan with an externally placed non‐standard filter 37 10. Doing oversize and batch scans 47


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1. Before you scan Here is a checklist of actions and checks that should be done either routinely or before starting a scan with the SkyScan 1172 micro‐CT scanner. Some of these items wil rl be eferred to again in more detail later in the document. 1.1. The scanner and its immediate vicinity should be in a clean condition and

clear of dust, particularly dust with any metallic or other content of dense material – such dust can potentially be problematic if it finds its way into the scanner, or onto scanned samples. Make sure that the air vents through which air is drawn in and blown out of the scanner, for cooling, are not obstructed. Don’t neglect to dust and clean the computer monitor, keyboard and mouse, and when necessary replace batteries of cordless components. Never( put a magnet anywhere on the scanner – e.g. don’t use a magnet tfix notes to the scanner!)

o

1.2. No part of the scanner should be in direct sunlight. Air conditioning is a good idea in the scanner room, particularly in hot weather.

1.3. The computer which controls the scanner should be kept in good working order. Keep at least 20% of the disc space free on the C drive (with the Skyscan control software), and at least 15% of other data disc drives. Perform defragmentation of all the hard disc drives every month or two. If the computer is internet‐connected, make sure Windows Updates are

omptly. installed pr1.4. Make sure flat field corrections are up to date for all scanner settings

which are in use. These should be refreshed routinely, at least once every two weeks. (It does no harm to take a new flat field at the start of each day’s

the alignment test ld be scanning). Less frequently but also regularly, shoudone every month or two.

1.5. Have a glance at the SkyScan website for any upgrades to the control software of your scanner. (RSS feeds are available to inform you of updates.) The same applies for reconstruction software NRecon and the analysis programs CT‐analyser and CT‐volume, as well as the other utilities such as DataViewer.

1.6. Remember that the micro‐CT scanner is a sensitive high‐precision instrument, and treat it accordingly. Insert and remove the sample holders at the stage with the minimum force possible, and never subject the scanner to jolts or strong vibrations. It is also not there to be leaned on, or used as a shelf for books, papers etc.


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2. Turning the scanner on and off 2.1. Turn on the scanner computer. 2.2. Turn the scanner on by clockwise rotation of the key (turn fully to “start”,

then let the key return to the vertical position, like when starting a car).

2.3. Turn on the SkyScan 1172 control software from the desktop icon. Wait for the various initialisations and progress bars (e.g. lifting, door, camera, rotation, filter, video) to run their course.

When startup is complete, the default control software screen will be shown:


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2.4. Click on the yellow circular x‐ray button under the top menu, to start

the x‐ray source. 2.5. When it is the first time in the day that the x‐ray source has been turned on,

then the process of “aging” will run. A progress bar will appear, showing the gradual increase in x‐ray source voltage and current. This will take 15 minutes to run. Aging takes place only the first time the source is turned on each day; after that, turn‐on will take only a few seconds. The purpose of aging is the protect the X‐ray source and prolong its working life.


urce to turn off after aging,

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2

You can choose whether or not to set the x‐ray sowith the tick box below the progress bar.

2.6. THE TIME SCHEDULE OF X_RAY SOURCE AGING The aging as described above will take place over 15 minutes, in the case that the source has not been on for of a period of up to 2 weeks. If the source has not been turned on for between 2 weeks and 2 months, then the source aging process will take 40 minutes. If the source has not been turned on for longer than 2 months, then the aging will take 2 hours. IT IS STRONGLY RECOMMENDED that you do not allow aging to take 40 minutes or 2 hours, by ensuring that the source is not left inactive for more than a week or two. This can be achieved by turning the source on every week at least once, and allowing the aging to run, even if the scanner is not being used. There are two reasons for this. One is that it is inconvenient for the user to have to wait 40 minutes or two hours before starting scanning. The other is that long periods of non‐activity are harmful to the micro‐focus x‐ray source, and can shorten its lifetime, causing failure of the source (requiring its expensive replacement) to occur sooner. Therefore it is recommended that the user turns the x‐ray source on routinely at least once per week. When the scanner is not being used, just wait until the aging has finished, then turn off the scanner.

.7. TURNING OFF THE SCANNER (a) It is recommended to remove any sample object from the scanner sample chamber (although the base can be left in place), and to close the scanner door, before turning off the scanner. (b) First turn off the scanner control software, either by the red cross in the top right corner of the software window, or under Actions menu / Exit (bottom of list). A progress bar will run briefly during scanner software shut‐down. Do not turn off the scanner with the key before the control software is fully shut down.


Please note: if any flat field corrections have been taken while the scanner has been on, then you will be prompted to save the flat fields in a configuration file. It is strongly recommended that you do save the configuration file as prompted. This issue is discussed in section 4. The saving of the configuration file will take up to half a minute, and the corresponding progress bar will be shown:

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(c) After the scanner control software is fully shut down, turn off the scanner with the key at the right end of the instrument.


(a) Resolutio

3. Refresh the flat field correction for the current cs anning mode (without changing settings)

3.1. When making a scan, it is best that a flat field correction has been taken for the scan mode no more than 2 weeks before the scan1. Look at an image of an empty field with no object (ambient image). Signs of an out‐of‐date flat field include: (a) Dark or light dots or lines in the ambient image; (b) Asymmetry of brightness in the image, i.e. one side brighter than the other; (c) Overall brightness a little too high or low. An ambient image with an up‐to‐date flat field correction should look like the image below, without any dots or lines and with uniform brightness. The profile line that is shown, is activated by right‐click anywhere on the image.

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3.2. A scanning “mode” means the selected combination of three scan parameters:

n level (small pixels 4k / medium pixels 2k / large pixels 1k).

1 Some users prefer to take a fresh flat field at the start of each day’s scanning, this is not a bad idea.


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(b) Filter (no filter / 0.5 mm aluminium / Cu + Al filter) (c) Camera position (near / mid / far). The camera position is set automatically by the scanner based on the user’s

to do anything about camera ent camera position is.

choice of pixel size: the user does not haveposition except be aware of what the curr

3.3. Camera position (c) is determined by the selected pixel size or magnification, and is shown in the bottom right corner of the 1172 control software screen (see image to the right). Pixel sizes in the central range correspond to the near camera position, while either very small pixels (high magnification) or very large pixels (low magnification) are acquired with the mid or far camera positions. Therefore it is important to note that a scanning mode is defined by the cam ze – era position, not the actual pixel sia single mode covers all the pixel sizes for that camera position.

3.4. To refresh the flat field correction for the current mode, you need an empty image. First take an image to check that no part of any object is present in the image. Objects can often be removed from the image field by moving them down – for instance set the Z position to zero. If the object is still visible in the image field, then open the scanner and remove the object. (It can help to go to the minimum pixel size for your current camera position – near, mid or far).

3.5. With an empty field obtained, wait until the x‐ray source has been on for 5‐10 minutes, as long as necessary to obtain stable output of the source. Left‐right asymmetry of image brightness can mean that the x‐ray source is not yet stable. Right‐click on the image to switch on the profile view, to assess symmetry of brightness (see the image in part 3.1). Note however, that if the flat field is out of date, then the ambient image might have asymmetrical brightness, even with the source stabilised. With the profile view is an indication in blue text of the minimum, maximum and average intensity value along the line. With the continuous video imaging on (video button), look at the “av” or average intensity value – this is 91.9% in the example image below. This value should remain about the same with some minor fluctuation, when the source is stable; if the value gradually increases or decreases, you must wait a little longer for the output to stabilise.


3.6. With the empty field obtained and the source stable, open the “acquisition modes” menu item under “options”.

3.7. The acquisition modes window shows you a table with the 27 available scanning modes (see the image at the end of this section). The current scanning mode is shown with an arrow sign:

In the complete acquisition modes window below (at the end of this section), the arrow sign shows that the currently selected mode is: far camera position, medium (2k) pixel size, and filter 1 (0.5mm aluminium).

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The camera exposure time for this mode is 2655 ms. 3.8. At the bottom of this acquisition modes window is a list of flat field

(“reference”) options to choose between. If you are scanning with the standard single image field (maximum image width 27‐35mm), then select the third option, “acquire bright+dark in central position for current mode”. However if you are using the left‐right offset imaging (maximum image width 50 or 68mm), then choose the 4th option, “acquire flat‐field reference for marked (*) modes. In this case, click on the current mode (i.e. on the

May 23, METHOD NOTES, SKYSCAN 1172 DESKTOP MICRO‐CT2008

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number 2655 in the above example) so that an asterisk * appears by the mode. Check that no other modes have an asterisk.

3.9. At the bottom of the window is an option: “X‐ray off after acquiring”. If the scanner is empty (no sample), then check this option – you will have to open the scanner after taking the flat field. But if the sample is in the scanner (but moved down out of the image field) then you can deselect the option, so that you can proceed with a scan after flat field acquisition is complete. Then click on “OK” and flat field acquisition will begin and proceed automatically.

Note ‐ if you want to take a fast flat field by correcting the bright field only (empty field with x‐rays on), then select the 2nd option, “acquire bright field in central position for current mode”. This will run faster than the 3rd option (bright + dark) and the source will not go off and thus will remain stable. However this option is not suitable for routine flat field refreshment – from time to time the dark field also needs to be refreshed.

3.10. Note finally: by selecting the 4th option, “acquire flat field reference for all marked modes”, multiple modes can be corrected in a batch operation. Clicking on any mode will add or remove an asterisk (*). Make sure that exposure times are correct for all modes (see chapter 6) before batch flat field correction.

May 23, 2 800 METHOD NOTES, SKYSCAN 1172 DESKTOP MICRO‐CT

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4. oi D4.1. Preparing your sample. A range of special sample holders are available

from SkyScan, such as vertical tubes of various sizes made of plastic or polystyrene foam, clip holders for very small or thin samples, hollow rod holders for particles or powders, and more. SkyScan users also have shown a lot of inventiveness and creativity in finding solutions themselves for mounting all manner of different samples. SkyScan is also open to requests for assistance in creating holder solutions for unusual types of sample. A few guidelines for mounting your sample for scanning: (a) The sample must be stable and secure. The smallest movement during a scan will severely compromise the results and require the scan to be repeated. Samples in vertical tubes must have sufficient contact with the tube walls to hold their vertical position by friction, so as not to slip down during a scan: paper tissue padding is useful for this. (b) The sample should be centered along the vertical rotation axis, as closely as possible. The accuracy of centering will increase the possible magnification, and will improve the geometric alignment of the scan. Poor centering and asymmetric rotation of the sample can cause the post‐alignment correction to fail, necessitating time‐consuming manual finding of the correct post‐alignment correction value during reconstruction. (c) No wet or moist object surfaces should be open to the air during a scan, such as soft biological tissue. Such objects should be wrapped in plastic film or placed in a closed plastic tube. Exposure of moist surfaces leads to drying and shrinkage during a scan, leading to movement artifacts and poor resultant images. Temperature can rise to well over 30 C inside the scanner during a scan, especially if the scan is of long duration. (d) There are air currents inside the scanner due to cooling fans, so loose, unsecured objects of light weight can flap around in the wind, or even be blown off the stage if unattached. Make sure the sample is secured with no loose parts. (e) Small objects allow higher magnification and resolution, and also minimise problems of beam hardening. Large objects by contrast require longer scans with smaller rotation steps, and sometimes more frame averaging, to overcome increasing image noise with sample size. Therefore, if you are scanning a homogeneous material to visualise and analyse its internal structure, keep to a minimum necessary the size of the sample, while scanning enough of it to see the structures of interest.

ng a scan


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(f) Batch scanning. Vertical tube sample holders, available from SkyScan, allow several samples to be loaded for scanning in automated batch mode. How to do batch or oversize scan is described in section 10. If batch scanning is possible, it is a good idea to do it, for a couple of reasons: firstly, it increases scan throughput and reduces the user workload; secondly, reducing the number of times the x‐ray is turned off and on will in the long term increase the lifetime of the x‐ray source.

4.2. With the sample and stage inserted into the screw base, carefully tighten the screw to finger tightness only – do not over‐tighten or apply strong force to the sample stage assembly.

4.3. Close the scanner door by clicking on the door icon at the top left of the control software window.

Wait for the process of door closing to be complete; the yellow circle x‐ray source button will change from grayed out to full colour . Then turn on the x‐ray source by clicking on the yellow source button.

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4.4. Go to the appropriate scan mode for your object. This means doing (or

checking) three things: (a) Select the correct filter, from the menu item Options / Filter. (b) Select the appropriate resolution level, small pixels 4k / medium pixels 2k / large pixels 1k. (c) Select the appropriate pi gnification). xel size (ma


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On closing the scanner and starting imaging, the object will by default be in the minimum magnification position, corresponding to the largest pixel size – see the upper of the two images below. You should increase the magnification – i.e. decrease the pixel size, until the object is at its maximum onscreen size without any part of it being outside the image field. Check at several rotation steps that the object is contained in the image field and rotating symmetrically.

Default minimum magnification, at 2k resolution level in this case 13.5 micron (11 Mpix camera).


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Optimised magnification, pixel size of 6.9 micron.

4.5. With the sample correctly positioned, with the right Z elevation of the sample as well as magnification, now click on the start scan button. Fill out the acquisition dialog for starting the scan. From the top, check that the pixel size is set correctly, here to medium pixels (2k). Give a filename prefix and set up a data directory by clicking on “browse”. Make your selections of rotation step, frame averaging and the other selectable options. Note – if you select partial width for a thin object, be aware that the rotation step displayed will change in response to restricting the image width – if you don’t want this change, write in your intended rotation step prior to scanning.

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4.6. The scan will start, giving a progress bar to indicate time remaining. If you

are intending a single scan only, it is advisable to select “x‐ray off after scanning”.

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5. The configuration files: a fast and easy way to change applied voltage

5.1. Adjusting the scanner to scan objects of lower or higher x‐ray density, requires the adjustment of energy or voltage of the x‐rays. The biggest changes to x‐ray energy are made by changing between the three filter settings, no filter for lowest energy, 0.5mm aluminium (Al) filter for higher, and the copper plus aluminium (Cu+Al) for the highest energy. This can be described as a “coarse adjustment” of x‐ray energy. Filters increase average x‐ray photon energy by selectively removing the lower energy x‐rays. Samples typically scanned with no filter include: soft biological tissue without calcification, animal embryos, plant tissue; also low density materials such as carbon fibre; scaffolds of low density such as PLA/PLGA bioscaffolds, collagen, hair, polyurethane, plastics of low density and small size. Samples typically scanned with 0.5 mm aluminium filter include: small bones (e.g. mouse), medium density materials, graphite, low density metals such as aluminium; lowmedium density rocks such as limestone, ceramics. Samples scanned with the Cu+Al filter include: larger bones (e.g. large mammal such as sheep), bones with metal implants, small metal samples, large and high density rock samples, small circuit boards. Filter is changed by going to the filter item under the options menu.

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5.2. However after choosing the appropriate filter, sometimes a “fine adjustment” of x‐ray energy is still needed, to further optimise image contrast. This can be achieved by adjusting the applied voltage. But changing the applied voltage for a given filter is more complex than just changing filter, which only requires one menu item selection. If the applied voltage is changed, it is imperative that three things are immediately done:


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(a) adjustment of the x‐ray camera exposure time, (b) taking of a new flat field correction, and (c) changing the information in the acquisition modes table. An experienced user will find these steps straightforward. However they may be daunting for a less experienced user, and for any user they take a little time to do. Therefore, the configuration files are provided as a quick and straightforward way to change applied voltage, avoiding the steps a, b and c listed above.

5.3. A configuration file contains all the settings information for all 27 of the scanning modes in the 1172 scanner, plus all the associated flat field correction images. In the Skyscan 1172 two configuration files are provided by default at installation, called std_high and std_low. These two configurations apply a high and a low voltage respectively to all three filter settings. The actual voltages set in these two configuration files are shown in the table below:

Configuration file Voltage for no filter

Voltage for 0.5 mm aluminium filter

Voltage for copper plus

um filter alumini

Std_High 60 kV 90 kV 100 kV

Std_Low 40 kV 50 kV 70 kV

5.4. When a SkyScan 1172 scanner is installed, typically the settings are set to the Std_High settings. So for example if the 0.5mm aluminium filter is selected, the applied voltage will go automatically to 90 kV. This is suitable for example for the scanning of rat bones. However if the user wishes to make a scan of mouse bones, then a more appropriate setting will be the same 0.5 mm Al filter but with 50 kV applied. To go to this setting, you need only to load the Std_Low configuration file: this simple procedure is set out below:

May 23, 2008 METHOD NOTES, SKYSCAN 11

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72 DESKTOP MICRO‐CT

5.5. Loading a configuration file: Go to the “Actions” menu and select

“Configurations”, the second to bottom item. Out of the sub‐options shown, choose “load”.

5.6. The configurations are stored in a subdirectory under the SkyScan scanner control software, called “config”: navigate to this directory in the file dialog box that opens after you select “load”, and you will see two options: Std_High and Std_Low. Choose Std_Low and click on “open”.

On clicking on “open”, a warning will appear asking you to confirm that you intend to replace all flat field images (“references”) and settings. Click “Yes”.

5.7. A progress bar will appear as the whole set of scan modes is updated with the low kV settings and flat field references. This will take a minute or so.


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5.8. An example of the set of acquisition modes obtained after loading the Std_Low configuration file is shown to the right. The scanner is ready to scan at these new, lower voltage settings. Note that the currently loaded configuration file, and its path, are shown in the top border of the acquisition modes window. Please note that loading a config file does not guarantee an up to date flat field. If the config has not been used for a while, it will be necessary to immediately refresh the flat field for your current scan mode.

May 23, 2008 METHOD NOTES, SKYSCAN 1172 DESKTOP

5.9. Finally,

MICRO‐CT

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three important points must be made: (a) The exposure times and settings will be correct in the loaded configuration file, but if the flat fields were collected several weeks or more ago, then the flat field will, as always, still need to be updated. The procedure for updating the flat field correction (but without changing settings of voltage, current or exposure time) is set out in part 1. (b) After a long period of a year or more of use of the scanner, it will be necessary to check that the exposure times for the scanning modes in the configuration files are correct. Small change in the x‐ray source output can necessitate change to the exposure time after long periods. The procedure for adjusting exposure time for a given scanner setting, is explained in section 4. (c) After updating one or more flat fields with a configuration, the configuration file needs to be resaved. Although flat field corrections are saved to disc automatically, they will be lost (overwritten) if any other configuration file is subsequently loaded. Again go to the Actions / configurations menu item, and choose save. Select the correct configuration file. A progress bar will appear until the saving is complete.

5.10. It should be noted that users can construct and save their own configuration files. This can contain any combination of voltages for the three filter options. For example the user may wish to set up a configuration with “medium” voltages, with intermediate values between the Std_Low and Std_High configurations. User configurations are useful where several people or groups are using a scanner, to keep ones carefully optimised and adjusted settings safely stored for re‐use.


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6. Adjusting the x-ray camera exposure time for a cs anning mode.

6.1. It is good practice to check from time to time the correctness of the exposure time for a scanning mode.

6.2. Go to an empty field (“ambient field”) by either moving the sample down out of the field of view, or removing the sample from the scanner. The ambient field, with flat field correction applied, might look like the image below.

This image shows a horizontal profile indicating uniform intensity with minor fluctuation, and an average value of near 90%. This is normal and reflects an up to date flat field. However it is important to note that a normal looking corrected image, does not automatically mean that the camera exposure time is optimal; an incorrect exposure can still result in a

normal corrected image similar to this. Therefore, the next step is to removethe flat field correction, and inspect the uncorrected or “raw” image.

6.3. To remove the flat field correction and view the raw, uncorrected image, go to the options menu and choose preferences.


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6.4. In the preferences window, the top item is “always snap image with flat field correction”; deselect this in order to see the raw, uncorrected image.

6.5. The uncorrected, raw image, shown below, has a very different appearance to flat‐field the corrected image. It is a darker shade of grey, and the profile line, in red (activated by right‐click of the mouse) shows less even brightness, as well as some bright or dark dots or lines. This is the image that the camera actually “sees”; the flat field correction converts the background to the more uniform and cleaner ambient image that you see with the flat field applied, and when scanning.


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looking corrected image might have a wrong exposure time, and vice versa. 6.6. Continuing the current example of the above raw image, the scanning mode

is: no filter (“filter 0”), medium 2k pixels, and the far camera position. To see the exposure time for this mode and adjust it, go to the options menu and select acquisition modes (see image below). In the acquisition modes table that opens up , the current scanning mode is shown by the arrow: for this mode the exposure time is 590 ms.

For adjusting exposure time, the important parameter to look at is the average image intensity, shown as “av”. The blue text gives you the minimum, maximum and average intensity of the profile line. In the raw image above the average intensity is 49.1%. This value of 49.1% is fine – you should aim for a value of close to 50%. Values in the range 40‐60% for average intensity are acceptable. Less than 40% and the signal to noise level becomes too low. At intensities above 80% the response of the camera becomes non‐linear, so to keep safely away from this region, an upper limit of 60% for the average intensity is recommended. 100% intensity represents saturation, which is bad. PLEASE NOTE: when considering the average intensity value, it is important to understand whether you are looking at the raw uncorrected image, or the flat field corrected image. The meaning of the intensity value is totally different in the two cases. Only in the uncorrected raw image does the average intensity tell you anything about the correctness or otherwise of the camera exposure time. As mentioned above, the appearance and average intensity of the corrected image will “hide” the reality, an OK

May 23, 2 800 METHOD NOTES, SKYSCAN 1172 DESKTOP MICRO‐CT

To change exposure time, click on the up and down arrow buttons (“spin buttons”) to the right of the box showing 590. Then click on “OK” at the bottom of the acquisition modes window.

6.7. Several ambient images are shown at the end of this section, which illustrate the effect of changing camera exposure time. (Please note: when you open the acquisition modes window, you see a large

d table of all 27 possible modes – combinations of filter, pixel size level ancamera position. The current mode is indicated with an arrow).

6.8. In the fastest scanning modes, such as at the large pixel (1k) resolution, near camera position and with no filter, you will need a very short exposure time. You will have noticed however that exposure time can be changed only in discreet intervals, not continuously. In these fastest modes the exposure time steps may be too large: neighboring exposure time values might be associated with too high and too low an intensity. In such a case, choose the lower intensity, not the higher, if necessary breaking the “rule” of the 40% lower limit. You can gain signal to noise ratio by choosing higher values of frame averaging during scans, in this case. With such fast image acquisition, large numbers of averaged frames per rotation step are

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possible. 6.9. A final note: please do not pay any attention to the absolute values of

exposure time given in this section or elsewhere in this method note, and try to apply these to your scanner. For the Skyscan 1172 scanner, there are several different models of x‐ray camera, for which exposure times will be significantly different for a given scanning mode. Even different cameras of the same model, will differ slightly in their sensitivity and thus an exposure time determined for one scanner is appropriate for that scanner alone. For the purpose of standardising scan parameters between scanners, it would be better to refer to the average intensity of the raw uncorrected image, as a standard point of reference, not the camera exposure time.


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Raw image (flat field deselected): camera exposure time reduced from 590 to 295 ms: average intensity is 27.5 % ‐ too low.

Raw image (flat field deselected): camera exposure time increased from 590 to 885ms: average intensity is 71.4 % ‐ too high.


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Raw image (flat field deselected): camera exposure time increased from 590 to 1475 ms: average intensity is 100%, the camera is SATURATED (this must be avoided, will cause corrupted images).

Raw image (flat field deselected): camera exposure time is set correctly to 590 ms. Intensity is 49.1% which is fine.


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7 anging the applied voltage 7.1. Go to an empty field (“ambient field”) by either moving the sample down

out of the field of view, or removing the sample from the scanner. Take the following example: we have selected the Cu+Al filter, and 90 kV (e.g. from the std_high configuration file). Also we are at the intermediate 2k resolution level (medium pixels). Camera is in the near position, with pixel size in the central range. We wish to change the applied voltage downwards, to 70 kV. With our empty, ambient field selected, first go to options / preferences and deselect the flat field, then view the raw, uncorrected image (as in steps 4.2‐4.5 above). The applied voltage is at the preset value of 90 kV. The raw image obtained is as shown below.

The average intensity is 53.0 %, which is in the acceptable range (40‐60%). Now the x‐ray applied voltage is lowered from 90 to 70 kV by the following steps: (a) Double‐click on the yellow triangular x‐ray source icon; the source power control window will open. (b) Move the slider for voltage, on the left, down from 90 to 70 kV. Note – the tick box at the bottom titled: “always maximise power” should by hecked.

. Ch

c


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Moving the slider will cause the blue bar to move to the required new voltage. With the tick box checked, the current blue bar will automatically adjust to a new value to maintain maximum source power. However to implement the chosen change of power setting, you then need to click on “apply”.

Now the red bars, indicating the actual power settings in the x‐ray source, will move to become equal to the red bars; the change of x‐ray source power settings is now complete. Close the source power control window.


7.2. With the x‐ray voltage decreased from 90 to 70 kV, again capture an image

of the ambient (empty) image with no flat field. The image obtained is shown below:

Now the average intensity has fallen to 31.7%, below the acceptable range

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7.4. Now edit the values of voltage and current for the Cu+Al filter in the acquisition modes window, changing them from 90 and 110 to 70 and 142 respectively. With the acquisition modes correctly updated, take a new flat field correction (bright and dark, 3rd or 4th option) – refer to part 2. It is advisable that the new settings and flat field correction are saved to a suitably named configuration file.

(40‐60%). 7.3. Now go to the acquisition modes (under the options menu). Go to the

current scanning mode (Cu+Al filter, 2k resolution level (medium pixels), camera in the near position) which is indicated by the arrow. The exposure time value is increased, using the up/down arrows, from 2400 to 4000 ms. (See the acquisition mode images on the next page below, the original values top, the changed values bottom. Again, the relevant mode is shown with the arrow.) Click OK to close the acquisition modes window. Take an ambient (uncorrected) image again: now the average intensity has increased to 55.2% ‐ an acceptable value.


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Initial table with 90 kV, and 2400 ms exposure time set.

Final table with 70 kV, and 4000 ms exposure time set. The voltage and current values in the appropriate boxes are changed (by manual editing) to 70 kV and 142 uA respectively.


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applied voltage. 8.1. Starting with a low density example, two projection images are shown

below, of polyethylene particle clusters in a plastic tube. The upper image is taken with no filter, and the lower image with the 0.5 mm Al filter (both with 40 kV applied voltage).

The lower image is not a mistake, it is of the same objects, but they are almost invisible. The Al filtration leaves only higher energy x‐rays, which are not absorbed in the low density sample. This is an example where correct filter choice is vitally important, such that you cannot even see your sample if the wrong filter option is selected.

8. Examples of improvements to projection image contrast and quality, achieved by adjusting filter and


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8.2. The second example in the series of images below, is of the head of a mouse. The filter selected in this case is the copper plus aluminium, with three different applied voltages.

The applied voltages in these three images, from top to bottom, are 100 kV, 70 kV and 50 kV respectively. Only the bottom image with 50 kV applied voltage has acceptable contrast, with a minimum transmission value of 22% and a significant part of the transmission profile below the 50% line. The upper two images are too pale, with insufficient absorption contrast.

100 kV

70 kV

50 kV


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8.3. The third example is a block of aluminium foam about 1.5 cm across. Aluminium has a density about the same as bone; depending on the sample size, Al foam can be scanned with either the 0.5mm Al filter or the Cu+Al filter. The first image, below, is an example of the projection image obtained with grossly insufficient filtration. The Al foam block is imaged with no filter. Transmission is much too low, with a minimum transmission of zero and the image visibly much too dark.

The images below (next page) show the results of using the two different filters, 0.5 mm Al and Cu+Al, each with a range of different applied voltages. Overall you should note the pattern of increasing contrast between high and low density with decreasing applied voltage. The recorded minimum transmission value, taken from the profile line of each image, is an indication of this. You will note also that there is overlap between the contrast of the images with the two filters. For instance, 70 kV with the 0.5 mm Al filter gives very similar appearance and transmission to 50 kV with the Cu+Al filter. Likewise 100 kV with the 0.5 mm Al filter gives a similar result to 70 kV with the Cu+Al filter. There is no simple rule that tells you what is the perfect or correct setting. From the six settings below, one can say that two settings are inappropriate: 50 kV with 0.5mm Al filter gives too dark an image, i.e. too little transmission, while 100 kV with the Cu+Al filter results in a projection image that is too pale, with insufficient contrast and absorption. Any of the other four settings would be acceptable. The 0.5mm Al filter settings will give significantly faster scans. However the use of higher filtration combined with lower applied voltage, although making the scan


time longer, has the effect of reducing beam hardening in the reconstructed images. This is because the spread of the energy distribution of the x‐rays is reduced. The maximum x‐ray energy is lower due to lower applied voltage, while the minimum x‐ray energy is higher, as a result of more filtration.

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Aluminium 0.5 mm filter, 50 kV. Copper plus aluminium filter, 50 kV. Minimum transmission 3.9 %. Minimum transmission 13.3 %

Aluminium 0.5 mm filter, 70 kV. Copper plus aluminium filter, 70 kV. Minimum transmission 11.4 %. Minimum transmission 23.5 %

Aluminium 0.5 mm filter, 100 kV. Copper plus aluminium filter, 100 kV. Minimum transmission 22.7 %. Minimum transmission 34.1 %.


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9. Setting up to scan with an externally placed non-ts andard filter

9.1. At the aperture leading to the camera on the left of the sample chamber is a slot into which the user can place any filter of their choice. This allows you to expand to filter options beyond the two available internal filters.

9.2. The present example will show how to set up the scanner to scan with 1mm

of aluminium filtration, at an applied voltage of 60 kV. 9.3. We will start at the 0.5mm aluminium filter setting (one of the two internal

filters) and at 50 kV applied voltage, the voltage assigned to this filter in the

“std_low” configuration setting (see chapter 5 for configuration files). 9.4. The object we will scan for this example is a piece of human cortical bone,

embedded in polymethyl methacrylate (PMMA) resin, shown in the image below.


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9.5. The need for an intermediate filter option is

indicated by the following two screen images from the 1172 scanner. These show projection images of the cortical bone sample first with the lower standard filter, the 0.5mm Al filter and 50 kV, and secondly with the high standard filter, the Cu+Al filterwith 70 kV (both these voltages are associated with the “std_low” configuration). Looking at the transmission percent values for both images, it is evident that the first image shows too little x‐ray transmission (the image is too dark), while the second image shows too much (the image is too pale).

9.6. We will therefore set up an intermediate setting, 1mm aluminium filtration with 60 kV. (The Cu+Al filter is equivalent approximately to 2mm Al filtration).

Projection image of the cortical bone sample with 0.5mm aluminium filter, 50 kV; transmission too low (minimum value 7.5%). Image too dark.


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Projection image of the cortical bone sample with copper plus aluminium filter, 70 kV; transmission too high (minimum value 25.5%). Image too pale. 9.8. Open the scanner door (x‐ray source will automatically turn off). Remove

the sample and stage. Take the 0.5mm aluminium filter made to fit the metal frame (these frames are supplied with the scanner – more can be obtained from SkyScan). Place the filter and frame into the camera aperture slot. Please note that there are two triangular notches in the underside of the filter frame, asymmetrically placed (see the images below). These correspond with notches in the filter slot, ensuring only one possible orientation for the filter, for consistent positioning.

9.9. With the filter in place, close the

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door and turn on the x‐ray source.9.10. Now change to applied voltage to

60 kV. Double click on the yellow triangular x‐ray power icon to open the x‐ray controls. Move the voltage slider to 60 kV, then click on “apply”. Keep the option “always maximise power” ch c ede k .


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The external 0.5 mm aluminium filter, adhered inside the metal filter frame, with symmetric notches on the lower side for positioning in the aperture. a

The external 0.5 mm aluminium filter fitted into the camera aperture slot.


9.11. Go to the resolution level and pixel size for the scan; in this case, to the

intermediate, 2k resolution level, and 5.2 micron pixel. Go to options / preferences, and deselect “always snap image with flat field”, so that you

will be looking at the raw, uncorrected background image with no sample. 9.12. Take an uncorrected image. Activate the profile line by right‐clicking on

the image near the centre. The image obtained in this case is shown below. The average intensity is now 34.5% ‐ too low a value. This is because of the increased filtration, from 0.5 to 1 mm aluminium.

Changing filtration from 0.5 to 1mm aluminum by adding a 0.5 mm Al external filter, results in uncorrected signal intensity to fall to 34.5% (even though applied voltage is increased from 50 to 60 kV). This must be compensated by increasing exposure ime – then taking a new flat field correction. t

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filter box to Al 1mm, and edit th

9.13. Now open the acquisition modes window (options menu). See the first window image below. The central filter column (filter 1) is still (now incorrectly) set as 0.5 mm Al filter, with applied voltage 50 kV. Edit the

7 e voltage and current boxes to 60 and 16(kV and uA respectively).

9.14. The mode box with the arrow indicating the current mode (near camera position, medium pixel size i.e. 2k) shows an exposure time of 885 ms. This is too low now due to the added filter. Increase the exposure time until an acceptable image intensity is obtained.


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The acquisition modes window before adjusting to the increase in filtration from 0.5 to 1mm aluminium. The arrow shows the current mode: near camera position, medium pixel size, 0.5mm Al filter. The exposure time is 885 ms. These settings need to be adjusted to those shown in the following image (below).


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m

The acquisition modes window after adjusting to the increase in filtration from 0.5 to 1mm aluminium. The arrow shows the current mode: near camera position, edium pixel size, 1 mm Al filter. The exposure time is now 1475 ms.


9.15. Following the change of settings shown above, including the increase in

exposure time from 885 to 1475 ms, an uncorrected image of the empty field is taken again. Now the raw intensity value is increased to 53.7 %, a good value. This acceptable value means we can now move on to take the new flat field correction.

9.16. To take the new flat field correction, go again to options, acquisition

modes. From the list of flat field reference options at the bottom, choose “acquire bright + dark in central position for current mode.

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9.17. Now the set‐up and correction of the new filter is complete. Now either

open the scanner and replace the sample – or, if the sample was small


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enough – just raise the vertical height of the sample back into the image field. Then go to options/preferences and turn back on the flat field correction. The new corrected image is shown below.

The projection image of the cortical bone sample with the 1mm filter (0.5mm internal plus the 0.5mm Al filter added externally), with voltage changed from 50 to 60 kV, and the settings correctly adjusted – including increase in exposure time – and the new flat field taken. This is now a better looking projection image, with a more suitable level of transmission. (Recall that transmission was too low with 0.5 m Al filter, but too high with Cu+Al filter). m

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1mm filter s

9.18. Now proceed to scan the sample at the new intermediate filter level of 1mm aluminium, which has now been correctly set up. (A crossection from the cortical bone scan with this

etup is shown to the right.)

9.19. Having gone to all this effort to set up this filter option, it is a good idea to save this configuration information by saving a new configuration file.


Chapter 5 gives full details about configuration files. Give the new config file a suitable name to indicate the special filter that has been set up. On clicking save, the save settings progress bar will appear – please wait till it completes.

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10 o. D ing oversize and batch scans 10.1. An oversize scan is a scan of an object over a height greater than the height

imaged in a single scan. Two or more scans are automatically made and connected together. Batch scanning means making several scans of different samples or sample regions, at several heights over the scannable range of about 7cm. A batch scan can include both single and oversize scans.

10.2. Load samples for scanning in one of the SkyScan tube sample holders for the 1172 scanner. Examples of such tube holders are shown in the figure below. Place them serially along the tube for scanning, taking care to

s record the identity of all samples. It helps to write the order of samplefrom top to bottom on a small piece of card or paper.

10.3. Place the tube holder in the scanner. Select the appropriate filter and scanner settings (for instance, load the appropriate configuration file). Turn on the x‐ray source and prepare for scanning in the usual way.

Vertical tube holders (left) for sample positioning and batch scanning in the SkyScan 172 scanner. Positioning of a holder in the scanner stage (right). 1 10.4. Go to the correct magnification, appropriate to achieve both the required

level of detail and also the necessary vertical extent of image from a single scan. Make sure that the scan mode corresponding to the chosen

y magnification – such as camera position (near, mid, far) has been recentlflat‐field corrected.

10.5. Now go to the actions menu and launch the oversize scan function – see image, below right.


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The vertical tube holder with mouse femur and BMD calibration rods, first at default inimum magnification (17.4 micron pixel, left) then at optimal magnification, with .8 micron pixel (right). m4 10.6. The scout view window which opens on the

“set oversize scan(s)” command can optionally be moved to the right of the displayed image. Then press “start” at the top of the scout window to begin the scout view of the whole vertical scan range. Wait for the scout view to finish, or stop the scout view when the samples of interest have all been imaged.

10.7. A series of images of the oversize / batch scout window are shown below, illustrating the set‐up of a batch scan consisting of a single oversize scan of the whole of a mouse femur, followed by two individual scans (single scan rotations), first of a pair of BMD calibration rods, then the whole of a mouse vertebra. The voxel size (nominal resolution) of all these scans is 4.8 um, the typical resolution for high throughput bone morphometry in the SkyScan 1172.


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• First click on start. (Left image.) The scout view images all objects in the scannable height range, the last of which is a whole mouse femur, as shown.

• Enter the sample prefix. The “top” button will become active. Click on it and then click on the position in the image, just above the top end of the femur,

be scanned. ( iddle image.) representing the upper limit of the range to M

• Then the “bottom” button becomes active. (Right image.)

• Note that whenever the mouse cursor is moved in the scout image window, a tool tip shows the z height – in the image to the left this z height is shown as 35.943 mm.


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single scan, the button “single scan” will become active.

• You can repeat left click to get the chosen area for a single scan just right. Then click on the “single scan” button. At this, the region scanned in a single scan will become highlighted, and the scan prefix again appears at the top of the selected range. Note that in the case of setting a single scan, you don’t have to specify the bottom limit, only the top. (Right image.)

• Click on the “bottom” button. Then click on the position in the scout image corresponding to the bottom of the scan range – here, just at the lower end of the proximal femur. At this, the selected range from top to bottom will become highlighted with a dot array, and the scan prefix “femur_whole_” will be shown at the top of the image range.

• Note that clicking on “bottom” finalises and completes the oversize scan; the scan prefix becomes visible on the scout view image but disappears from the “prefix” box at the top left of the window. This box is now ready for the entry of the next scan. (Left image.)

• Move up the scout view range by clicking on the down arrow on the scroll bar to the right. The next objects moving upwards are a pair of BMD calibration rods. (Middle image. A single scan encompassing both rods will suffice for density calibration.)

• Enter the prefix in the box, here “BMDcal_rods_”. At first only the “top” button is active. However, if you left click at the top of the field that you require for a


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• After setting up the BMD rod scan, the prefix window again becomes empty. Now scroll up to the next sample, a whole mouse vertebra. Enter the prefix, here “vertebra_”. Again, left click to denote the required top

tive. margin of the scanned area. The “single scan” button will become ac

• Click on “single scan”. Once again, the scan of the vertebra becomes finalised, the scanned region is highlighted with a dot array, the scan name appears in the scout image, and disappears from the prefix box.

• This three‐sample batch scan, comprising an oversize scan (whole mouse femur) and two single scans (BMD rod pair and mouse vertebra) is now prepared.

• To move to launching the batch scan, click on the “start scan” button. button which is connected y after the scan is complete.

(Note – don’t confuse this with the “start now”with auto‐launching of reconstruction, but onl


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10.8. With the batch scan pre‐programmed as just described, in the scout view

dialog window, a standard scan (“acquisition”) window will open. First you must set a single directory for all the batch scan projection images to be written to. Note however that when subsequently reconstructing the scans in NRecon, you can reconstruct each scan to a separate and separately named directory.

e 10.9. Check that the scan parameters, of rotation step, frame averaging, etc., arcorrectly set. Then click on “OK” to launch the batch scan.

10.10. The scan will start at the bottom end of the stack of samples. In the case that the scan is an oversize scan, the scan will move from bottom upwards – in this case the scan of the whole mouse femur is commencing at the lower, proximal end (see image below).


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The batch scan commences with the lowest sample, and in the case of an oversize scan – here of the whole of a mouse femur, scanning starts at the lower margin, in this case at the proximal end. As in all scans, scan progress is indicated by a progress ar. b

Documents

Method Notes 1172