Improved digital SEM of cancellous bone: scanning direction of detection, through focus for in-focus and sample orientation

Three-dimensional (3D) study of cancellous bone tissue organization is necessary to understand how modelling and remodelling processes regulate bone structure and connectivity. It requires imaging methods that have both sufficient resolution power and width and depth of field. Since clinical imaging methods fall far short of the first requirement, we can only study prepared tissue in isolation from the body. Scanning electron microscopy (SEM) of macerated plane parallel slices is the most productive method, but we meet special technical problems in imaging porous bone because samples need to be relatively thick to maintain both continuity and context. Problems due to charging under the electron beam can be controlled by imaging with only high-energy backscattered electrons (BSE). This gives an important additional benefit that the direction of apparent illumination can be manipulated by positioning the detector, and multiple detector positions can be employed strategically to generate images in which colour is used to help in coding surface morphology. However, we next confront the difficulty of the limited depth of field. This can be improved by taking series of images, moving the sample along the electron optic axis, and combining these to generate a single extended-focus image. SEM imaging geometry gives a change in magnification with change of working distance, and it is shown that this must be corrected for each image of the through-focus sequence. Colour coding the lighting direction and increasing the depth of field are approaches that can be combined, and are well matched to the possibilities offered by communication by digital data projection. Finally, the latter means also offer another powerful technique for 3D representation through the display of through tilt image sequences. The novel routines considered here are generally applicable to all classes of microanatomical SEM sample.