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We developed here an iodine-containing probe that can be used to identify the molecules of interest in secondary ion mass spectrometry (SIMS) by simple immunolabelling procedures. The immunolabelled iodine probe was readily combined with previously-developed SIMS probes carrying fluorine, to generate dual-channel SIMS data. This probe should provide a useful complement to the currently available SIMS probes, thus expanding the scope of this technology.

Nanoscale imaging with the ability to identify cellular organelles and protein complexes has been a highly challenging subject in the secondary ion mass spectrometry (SIMS) of biological samples. This is because only a few isotopic tags can be used successfully to target specific proteins or organelles. To address this, we generated gold nanoprobes, in which gold nanoparticles are conjugated to nanobodies. The nanoprobes were well suited for specific molecular imaging using NanoSIMS at subcellular resolution. They were demonstrated to be highly selective to different proteins of interest and sufficiently sensitive for SIMS detection. The nanoprobes offer the possibility of correlating the investigation of cellular isotopic turnover to the positions of specific proteins and organelles, thereby enabling an understanding of functional and structural relations that are currently obscure.

Electron microscopy (EM) has been employed for decades to analyze cell structure. To also analyze the positions and functions of specific proteins, one typically relies on immuno-EM or on a correlation with fluorescence microscopy, in the form of correlated light and electron microscopy (CLEM). Nevertheless, neither of these procedures is able to also address the isotopic composition of cells. To solve this, a correlation with secondary ion mass spectrometry (SIMS) would be necessary. SIMS has been correlated in the past to EM or to fluorescence microscopy in biological samples, but not to CLEM. We achieved this here, using a protocol based on transmission EM, conventional epifluorescence microscopy and nanoSIMS. The protocol is easily applied, and enables the use of all three technologies at high performance parameters. We suggest that CLEM-SIMS will provide substantial information that is currently beyond the scope of conventional correlative approaches., Competing Interests: The authors have declared that no competing interests exist.

The cellular functions of lipids in the neuronal plasma membranes have been increasingly acknowledged, particularly their association to neuronal processes and synaptic plasticity. However, the knowledge of their regulatory mechanisms in neuronal cells remains sparse. To address this, we investigated the lipid organization of the plasma membranes of hippocampal neurons in relation to neuronal activity using secondary ion mass spectrometry imaging. The neurons were treated with drugs, particularly tetrodotoxin (TTX) and bicuculline (BIC), to induce chronic activation and silencing. Distinct lipid organization was found in the plasma membrane of the cell body and the neurites. Moreover, significant alterations of the levels of the membrane lipids, especially ceramides, phosphatidylserines, phosphatidic acids, and triacylglycerols, were observed under the TTX and BIC treatments. We suggest that many types of membrane lipids are affected by, and may be involved in, the regulation of neuronal function.

Boron has been employed in materials science as a marker for imaging specific structures by electron energy loss spectroscopy (EELS) or secondary ion mass spectrometry (SIMS). It has a strong potential in biological analyses as well; however, the specific coupling of a sufficient number of boron atoms to a biological structure has proven challenging. Herein, we synthesize tags containing closo-1,2-dicarbadodecaborane, coupled to soluble peptides, which were integrated in specific proteins by click chemistry in mammalian cells and were also coupled to nanobodies for use in immunocytochemistry experiments. The tags were fully functional in biological samples, as demonstrated by nanoSIMS imaging of cell cultures. The boron signal revealed the protein of interest, while other SIMS channels were used for imaging different positive ions, such as the cellular metal ions. This allows, for the first time, the simultaneous imaging of such ions with a protein of interest and will enable new biological applications in the SIMS field., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)