Quantitative X-ray imaging on bones using Faxitron and ImageJ


  •  Faxitron MX20 and digital imaging system
  • Calibration standards: 1-mm steel wire (7.9 g/cm³); 1-mm diameter spectographically pure aluminium wire (2.7 g/cm³); 1-mm diameter polyester fibre ( 1.4 g/cm³).
  • ImageJ software (1.43n)
  • Adobe Photoshop (7.0.1 or later)


Sample preparation

  1. Euthanise mice. Mice can be any age from 4 weeks onwards.
  2. Remove skin from carcass
  3. Upper limb: Cut under the scapula with a scalpel to detach from the thorax, but through the clavicle as close to the sternum as possible
  4. Lower limb: Cut vertically medial to the pelvis and lateral to the vertebrae in order to avoid the femoral head.
  5. Proximal six tail vertebrae: Remove the skin, cut the tail at the base. Count down inter-vertebral spaces and detach the six most proximal vertebrae.
  6. Place limbs and tail vertebare in 70% ethanol in 15 ml falcon tubes. Store at 4°C for at least 48 h.
  7. Dissect muscle and soft tissue away. Detach humerus from the scapula and femur from the acetabulum. Place bones in a falcon tube containing fresh 70% ethanol and store at 4°C.


Faxitron digital X-ray imaging

  1. Switch on Faxitron machine at least 15 min prior to use.
  2. Calibration: Remove sample stage. Calibrate by running four flat dark field images using time set at 15 s and voltage at 26 kV, save the calibration settings. 
  3. Remove bones from the ethanol solution and place on a paper towel to remove excess ethanol. Arrange them on the sample tray along with the steel, aluminium, and polyester calibration standards.

    Long bones of the hind limbs and tail vertebrae aligned with an aluminium standard prior to imaging.

  4. Insert sample tray in to x5 magnification uppermost slot in the Faxitron machine and close the door.
  5. Deselect Auto Level, Auto Exposure Control, Contrast Assist and Sharpen assist toggles.
  6. “Start Exposure” to commence imaging.
  7. Save image as a DICOM file.


Image processing (ImageJ)

  1. Open DICOM image in ImageJ. Press “h” to show histogram with the distribution of grey scale pixels within the image.
  2. Record minimum and maximum grey level values.
  3. Use rectangular Selections Tool to select as large an area as possible of the polyester standard and press “h”. Note value of modal grey level. Repeat this for the steel standard, but not for the aluminium standard which is an internal reference for the following grey scale stretch procedure.
  4. Stretch the image to the modal grey level values of the plastic and steel standards (Image > Adjust > Brightness/Contrast). Set minimum to the plastic modal grey level and maximum to the steel modal grey level.
  5. Convert to 8-bit (Image > Type > 8-bit). The resulting image has 256 grey levels, the plastic and steel standards are assigned grey levels of 0 and 255, respectively. Save in TIFF format.
  6. Invert image (Edit > Invert).
  7. Generate pseudo-coloured images: Open stretched 8-bit grey scale TIFF image in ImageJ. Go to Image > Lookup Tables > 16 colours. Save as a TIFF file.



Image processing (Photoshop)

  1. Open pseudo-coloured image in Photoshop.
  2. Select the brush tool, adjust brush size to 33 px diameter, select black as the brush colour.
  3. Zoom in to 200% and use brush to black out any soft tissue beyond the edge of the bone. If in doubt, it is better to remove a small bit of bone rather than leaving areas of soft tissue because this would skew the mineralisation data.
  4. Select the magic wand tool and click adjacent to the first bone. A yellow line should appear around the bone. If edges are not clearly highlighted, black out any soft tissue remaining. Repeat this for every bone. ( Different procedure for the tail vertebrae: Select pencil tool with a diameter of 13 px with 100% hardness. Zoom to 300% and draw a line around individual vertebrae. Select paint bucket tool and fill the background outside the vertebrae with black.
  5. Save cleaned image as a new TIFF file with the following settings: Image Compression: NONE; Byte Order: Macintosh.


Determination of relative mineral content (ImageJ)

  1. Open cleaned file in ImageJ. Go to Plugins > Macro > Install and open CustomHistogram.txt. You may have to add this file to the Macro folder where Image J is installed first.
  2. Select all bones to be analysed using the Wand (tracing) tool whilst holding down the shift key. You have to click right next to the bone you want to select. Holding the alt key and clicking removes areas from the selection.
  3. Select Plugins > Macro > CustomHistogram. Select following values: X Min:0, X Max: 255, Y Max: auto, Bins: 16 and press OK.
  4. Press “List” button in the displayed histogramm. Copy “bin start” and “count” columns into an Excel spreadsheet. These are the number of pixels in each of the 16 equal grey level divisions (bins) to give a frequency of grey level distribution for the whole image.
  5. Calculate the relative frequencies and cumulative frequencies of the grey levels.
  6. Statistical analysis of difference between cumulative frequency distribution of the grey levels of wild-type versus mutant bones can be performed using a Kolmogorov-Smirnov test on each of the grey scale images to determine if they differ from one another.



 J.H.D. Bassett, A. van der Spek, A. Gogakos, et al. , Methods in molecular biology (Clifton, N.J.) (vol. 816)