TyrRu: A Tyramide-Amplified Multimodal Ruthenium Tag for Elemental Mass Spectrometry and Immunofluorescence Imaging

Tyramide signal amplification, used in immunohistochemistry to image low-abundance biomolecules, has seen adaptation beyond conventional microscopy and is utilized in flow cytometry and electron microscopy. Here, we applied tyramide signal amplification to image biomolecules via elemental mass spectrometry imaging (MSI). Elemental MSI techniques such as laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) are used to image biomolecules via metal-conjugated probes, and resolution is typically hampered by a low detection sensitivity. We report the synthesis of a bespoke ruthenium complex (TyrRu) for tyramide amplification and describe its application as a multimodal analyte to image and quantify glial fibrillary acidic protein in mouse brain by complementary immunofluorescence and elemental MSI. Pairing TyrRu and avidin-biotin-HRP amplification produced a signal-to-noise ratio 2× higher than that obtained with a commercial metal-chelating polymer, allowing LA-ICP-MS image resolution of 1 μm. Data obtained using immunofluorescence and LA-ICP-MS showed excellent biomolecule agreement (r = 0.86). TyrRu was also applied alongside primary-conjugated antibodies in a multiplexed analysis, and the enhanced detection sensitivity facilitated the generation of an image with submicrometer resolution after super-resolution reconstruction.