Maxim B. Prigozhin

Cathodoluminescence (CL) microscopy has the potential to achieve a key goal in biological imaging: the simultaneous visualization of proteins and cellular ultrastructure. This goal can be attained by tagging proteins of interest with spectrally distinct cathodoluminescent probes for detection in electron microscopy. To this end, lanthanide nanoparticles (LNPs) are promising probe candidates due to their stability under the electron beam and their distinct ion-dependent emission spectra suitable for multiplexed detection. However, the hydrophobic surface chemistry of LNPs limits their use in biological samples and requires surface functionalization compatible with aqueous environments and EM sample preparation protocols. Here, we use a DNA-based ligand exchange strategy that renders cathodoluminescent LNPs hydrophilic and compatible with further functionalization for specific protein labeling. We characterize the CL emission of DNA-functionalized LNPs following aqueous transfer and common EM preparation steps, including osmium tetroxide staining and drying protocols based on hexamethyldisilazane and critical point drying, and show that LNPs retain their CL emission under all tested conditions. Finally, we demonstrate multicolor CL imaging of spectrally distinct, DNA-functionalized LNPs on the surface of mammalian cells, enabling simultaneous visualization of cellular ultrastructure via secondary electrons and LNPs via multiple CL color channels.

Cathodoluminescence (CL) microscopy offers a promising approach to nanoscale analysis, enabling detection of optical emission from a sample while leveraging the high resolution of electron microscopy. However, achieving multicolor single-particle CL imaging remains a significant challenge. Here, using lanthanide nanoparticles as a model system, we identify a critical limitation in CL imaging: nonlocal signal caused by stray electrons. We mitigate these nonlocal excitations and demonstrate multicolor single-particle CL imaging of nanoparticles down to 12 nm in diameter. Using this enhanced sensitivity, we demonstrate that CL brightness increases monotonically with nanoparticle diameter. We propose that multicolor imaging of spectrally distinct nanoparticles in the same field of view, coupled with the scaling of CL brightness with nanoparticle size, is crucial for confirming single-particle CL detection. Finally, we demonstrate the utility of our findings by imaging lanthanide nanoparticles in a biological sample. This work advances our understanding of nanoscale photonic responses to free electrons, establishing CL as a useful contrast mechanism for high-resolution, multicolor electron microscopy. Electron-induced light, cathodoluminescence, enables nanoscale optical analysis across disciplines. Here, the authors achieve multicolor cathodoluminescence imaging of sub-15-nm lanthanide nanoparticles and demonstrate its application for bioimaging.

Intermolecular interactions underlie all cellular functions, yet visualizing these interactions at the single-molecule level remains challenging. Single-molecule localization microscopy (SMLM) offers a potential solution. Given a nanoscale map of two putative interaction partners, it should be possible to assign molecules either to the class of coupled pairs or to the class of noncoupled bystanders. Here, we developed a probabilistic algorithm that allows accurate determination of both the absolute number and the proportion of molecules that form coupled pairs. The algorithm calculates interaction probabilities for all possible pairs of localized molecules, selects the most likely interaction set, and corrects for any spurious colocalizations. Benchmarking this approach across a set of simulated molecular localization maps with varying densities (up to ∼55 molecules μm −2 ) and localization precisions (1 to 50 nm) showed typical errors in the identification of correct pairs of only a few percent. At molecular densities of ∼5 to 10 molecules μm −2 and localization precisions of 20 to 30 nm, which are typical parameters for SMLM imaging, the recall was ∼90%. The algorithm was effective at differentiating between noninteracting and coupled molecules both in simulations and experiments. Finally, it correctly inferred the number of coupled pairs over time in a simulated reaction–diffusion system, enabling determination of the underlying rate constants. The proposed approach promises to enable direct visualization and quantification of intermolecular interactions using SMLM.

Intermolecular interactions underlie all cellular functions, yet visualizing these interactions at the single-molecule level remains challenging. Single-molecule localization microscopy (SMLM) offers a potential solution. Given a nanoscale map of two putative interaction partners, it should be possible to assign molecules either to the class of coupled pairs or to the class of non-coupled bystanders. Here, we developed a probabilistic algorithm that allows accurate determination of both the absolute number and the proportion of molecules that form coupled pairs. The algorithm calculates interaction probabilities for all possible pairs of localized molecules, selects the most likely interaction set, and corrects for any spurious colocalizations. Benchmarking this approach across a set of simulated molecular localization maps with varying densities (up to ∼ 50 molecules µm − 2 ) and localization precisions (5 to 50 nm) showed typical errors in the identification of correct pairs of only a few percent. At molecular densities of ∼ 5-10 molecules µm − 2 and localization precisions of 20-30 nm, which are typical parameters for SMLM imaging, the recall was ∼ 90%. The algorithm was effective at differentiating between non-interacting and coupled molecules both in simulations and experiments. Finally, it correctly inferred the number of coupled pairs over time in a simulated reaction-diffusion system, enabling determination of the underlying rate constants. The proposed approach promises to enable direct visualization and quantification of intermolecular interactions using SMLM.

Abstract Electron microscopy (EM) and fluorescence imaging are indispensable techniques that provide complementary information on cellular organization. Combining these two modalities is a long-standing challenge in bioimaging. In principle, it should be possible to use the electron beam both for ultrastructural imaging and for molecular localization. The latter could be accomplished by directly exciting suitable biomolecular labels and detecting their luminescence – a process termed cathodoluminescence (CL). Here, we achieve multicolor, single-particle CL imaging of sub-20-nm lanthanide nanocrystals (cathodophores) in the same field of view on the surface of a mammalian cell while simultaneously imaging cellular ultrastructure. In pursuit of this goal, we have developed a comprehensive framework for single-particle CL imaging of lanthanide nanocrystals. By mitigating nonlocal excitation due to secondary electrons, we achieved single-particle detection of multiple spectrally distinct types of sub-20-nm cathodophores. The smallest detectable cathodophores were sub-12 nm in diameter. We found that the CL emission rate scaled linearly with nanocrystal diameter. Furthermore, even in the absence of inert shells, cathodophores were not quenched in the context of mammalian cells processed for EM imaging using heavy-metal staining and sputter-coating. These findings establish cathodophores as promising biomolecular tags for multicolor EM. Moreover, our results inform general design rules for precise control and rational engineering of future generations of single-particle cathodoluminescent nanoprobes.

Glucagon signaling is essential for maintaining normoglycemia in mammals. The arrestin fold superfamily of proteins controls the trafficking, turnover, and signaling of transmembrane receptors as well as other intracellular signaling functions. Further investigation is needed to understand the in vivo functions of the arrestin domain-containing 4 (ARRDC4) protein family member and whether it is involved in mammalian glucose metabolism. Here, we show that mice with a global deletion of the ARRDC4 protein have impaired glucagon responses and gluconeogenesis at a systemic and molecular level. Mice lacking ARRDC4 exhibited lower glucose levels after fasting and could not suppress gluconeogenesis at the refed state. We also show that ARRDC4 coimmunoprecipitates with the glucagon receptor, and ARRDC4 expression is suppressed by insulin. These results define ARRDC4 as a critical regulator of glucagon signaling and glucose homeostasis and reveal a novel intersection of insulin and glucagon pathways in the liver.