Biology subjects

Prigozhin, M. B.

Publications and source records attributed to Prigozhin, M. B..

2 recordsLinked to original sources

bioRxiv2024

Identifying Intermolecular Interactions in Single-Molecule Localization Microscopy

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 {micro}m-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 {micro}m-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.

biophysics↗

bioRxiv2023

Lanthanide Cathodophores for Multicolor Electron Microscopy

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.

biophysics↗