Sample preparation and Imaging
Mice were injected with Calcein 5 days and 2 days before killing. Tibia and vertebrae were fixed in phosphate buffered formalin, embedded in methylmethacrylate and the blocks sectioned at 5µm.
For measurement of trabecular architecture and osteoid, sections were stained with van Gieson/von Kossa, and for analysis of resorption parameters with Aniline Blue and TRAP. Calcein Blue was used as a fluorescent bone stain that does not affect calcein labels, and was visualised using a DAPI fluorescence filter set. For details of the processing and staining methods: Link to zip file with protocols
To obtain images with sufficient resolution, Trap and van Gieson/von Kossa stained slides normally require to be imaged with a 10x objective lens (resolution of 1-1.5 µm/pixel). The Calcein double label require a higher resolution (<0.5 µm) and is usually imaged using a 20x objective. The undecalcified bone sections are rarely perfectly flat, and therefore using a extended depth of field imaging method helps to keep (especially calcein double labels) things in full focus. The StackFocusser Plugin (available from ImageJ website) does a good job. A slightly modified version (faster through use of multithreading) is available from me, please email email@example.com.
The most efficient method for imaging the slides is by using a slide scanner, such as those used in Pathology departments. Using a fully motorised microscope also works quite well. If these systems are not available, sections need to be imaged using several field, and the resulting images stitched together to create suitable input images for the software. This can be done using the “Stitching” plugin in the Fiji distribution of ImageJ, or using the free Microsoft Image Composite Editor (ICE) software, available from http://research.microsoft.com/en-us/um/redmond/groups/ivm/ice/. The Microsoft program is the easier one to use.
I have developed a few ImageJ macros that help with acquiring suitable images for the software using a manual microscope. These macros are written for use with QImaging cameras (which can be controlled through ImageJ). The macros can be adapted for other systems. Macros available on request by email.
TRAP-Aninline Blue stain
|LEFT:Mouse lumbar vertebra. Imaged using Zeiss AxioImager with 10x objective, and QImaging Retiga camera. Image stitched from 9 fields. Extended depth of focus using StackFocusser plugin from 5 focus levels.|
van Gieson/von Kossa stain
Human bone biopsy. Imaged using Zeiss AxioImager with 10x objective, and QImaging Retiga camera. Stitched from >30 image fields
Calcein double label with Calcein Blue counterstain.
|LEFT: Imaged using Zeiss fully motorised Axioplan microscope with 20x objective, DAPI and FITC filtersets.|