X-rays have been used for non-invasive high-resolution imaging of thick biological specimens since their discovery in 1895. They are widely used for structural imaging of bone, metal implants, and cavities in soft tissue. Recently, a number of new contrast methodologies have emerged which are expanding X-ray’s biomedical applications to functional as well as structural imaging. These techniques are promising to dramatically improve our ability to study in situ biochemistry and disease pathology. In this review, we discuss how X-ray absorption, X-ray fluorescence, and X-ray excited optical luminescence can be used for physiological, elemental, and molecular imaging of vasculature, tumours, pharmaceutical distribution, and the surface of implants. Imaging of endogenous elements, exogenous labels, and analytes detected with optical indicators will be discussed.

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