<p>In situ imaging of proteins, RNA, immune cells and other biomolecules is necessary to determine their function, interactions and roles in disease pathology. Increasingly, this is achieved via metal-conjugated probes in conjunction with elemental mass spectrometry imaging (MSI). This targeted technique is capable of simultaneously imaging up to 40 analytes, in comparison to the traditional bioimaging techniques that use fluorescent or chromogenic reagents that are typically restricted to less than four analytes without complex sample handling and analysis workflows. These analyses, however, are not straightforward, with a number of factors that require optimization. They require the use of probes specific to the target biomolecules, which are conjugated with analytes detectable by elemental MSI. Here, we summarize the MSI technology, the types of biological probes used for identification, and the forms of metal analytes used. We provide examples of their application including understanding cancer cell heterogeneity to direct clinical trials, which may impact clinical diagnostics and personalized medicine. We conclude with future perspectives on the potential of the technique and what is required to meet it.</p><p></p>