Boron-Containing Probes for Non-optical High-Resolution Imaging of Biological Samples.

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.

X10 expansion microscopy enables 25-nm resolution on conventional microscopes.

Expansion microscopy is a recently introduced imaging technique that achieves super-resolution through physically expanding the specimen by ~4×, after embedding into a swellable gel. The resolution attained is, correspondingly, approximately fourfold better than the diffraction limit, or ~70 nm. This is a major improvement over conventional microscopy, but still lags behind modern STED or STORM setups, whose resolution can reach 20-30 nm. We addressed this issue here by introducing an improved gel recipe that enables an expansion factor of ~10× in each dimension, which corresponds to an expansion of the sample volume by more than 1,000-fold. Our protocol, which we termed X10 microscopy, achieves a resolution of 25-30 nm on conventional epifluorescence microscopes. X10 provides multi-color images similar or even superior to those produced with more challenging methods, such as STED, STORM, and iterative expansion microscopy (iExM). X10 is therefore the cheapest and easiest option for high-quality super-resolution imaging currently available. X10 should be usable in any laboratory, irrespective of the machinery owned or of the technical knowledge.

A contamination-insensitive probe for imaging specific biomolecules by secondary ion mass spectrometry.

Imaging techniques should differentiate between specific signals, from the biomolecules of interest, and non-specific signals, from the background. We present a probe containing (15)N and (14)N isotopes in approximately equal proportion, for secondary ion mass spectrometry imaging. This probe designed for a precise biomolecule analysis is insensitive to background signals.

Secondary-ion mass spectrometry of genetically encoded targets.

Secondary ion mass spectrometry (SIMS) is generally used in imaging the isotopic composition of various materials. It is becoming increasingly popular in biology, especially for investigations of cellular metabolism. However, individual proteins are difficult to identify in SIMS, which limits the ability of this technology to study individual compartments or protein complexes. We present a method for specific protein isotopic and fluorescence labeling (SPILL), based on a novel click reaction with isotopic probes. Using this method, we added (19) F-enriched labels to different proteins, and visualized them by NanoSIMS and fluorescence microscopy. The (19) F signal allowed the precise visualization of the protein of interest, with minimal background, and enabled correlative studies of protein distribution and cellular metabolism or composition. SPILL can be applied to biological systems suitable for click chemistry, which include most cell-culture systems, as well as small model organisms.