Long-Term, Single-Molecule Imaging of Proteins in Live Cells with Photoregulated Fluxional Fluorophores.

Single-molecule localization microscopy (SMLM) can reveal nanometric details of biological samples, but its high phototoxicity hampers long-term imaging in live specimens. A significant part of this phototoxicity stems from repeated irradiations that are necessary for controlled switching of fluorophores to maintain the sparse labeling of the sample. Lower phototoxicity can be obtained using fluorophores that blink spontaneously, but controlling the density of single-molecule emitters is challenging. We recently developed photoregulated fluxional fluorophores (PFFs) that combine the benefits of spontaneously blinking dyes with photocontrol of emitter density. These dyes, however, were limited to imaging acidic organelles in live cells. Herein, we report a systematic study of PFFs that culminates in probes that are functional at physiological pH and operate at longer wavelengths than their predecessors. Moreover, these probes are compatible with HaloTag labeling, thus enabling timelapse, single-molecule imaging of specific protein targets for exceptionally long times.

Inversion of Configuration at the Phosphorus Nucleophile in the Diastereoselective and Enantioselective Synthesis of P-Stereogenic syn-Phosphiranes from Chiral Epoxides.

Nucleophilic substitution results in inversion of configuration at the electrophilic carbon center (SN 2) or racemization (SN 1). The stereochemistry of the nucleophile is rarely considered, but phosphines, which have a high barrier to pyramidal inversion, attack electrophiles with retention of configuration at P. Surprisingly, cyclization of bifunctional secondary phosphine alkyl tosylates proceeded under mild conditions with inversion of configuration at the nucleophile to yield P-stereogenic syn-phosphiranes. DFT studies suggested that the novel stereochemistry results from acid-promoted tosylate dissociation to yield an intermediate phosphenium-bridged cation, which undergoes syn-selective cyclization.

Polarity Sensitive Bioorthogonally Applicable Far-Red Emitting Labels for Postsynthetic Nucleic Acid Labeling by Copper-Catalyzed and Copper-Free Cycloaddition.

Two series of new, water-soluble, membrane-permeable, far-red/NIR emitting benzothiazolium-based fluorescent labels with large Stokes' shifts were synthesized that can be conjugated to alkyne-modified biomolecules through their azide moiety via azide-alkyne cycloaddition. We have used these azide bearing labels to make fluorescent DNA constructs using copper-catalyzed "click" reaction. All dyes showed good or remarkable fluorescence intensity enhancement upon conjugation to DNA. We also investigated the possibility to incorporate the benzocyclooctyne motif through rigid (ethnynyl) or flexible (ethyl) linkers into the DNA, thus enabling copper-free labeling schemes. We observed that there is a marked difference between the two linkers applied in terms of optical properties of the labeled oligonucleotides. We have also tested the in vivo labeling potential of these newly synthesized dyes on HeLa cells previously transfected with cyclooctynylated DNA. Confocal fluorescent images showed that the dyes are all able to cross the membrane and suitable for background-fluorescence free fluorescent tagging of nucleic acids. Moreover, we have observed different accumulation of the two dye series in the endosomal particles, or in the nuclei, respectively.

Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells.

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

Rapid Characterization and Quantification of Extracellular Vesicles by Fluorescence-Based Microfluidic Diffusion Sizing.

Extracellular vesicles (EVs) are emerging as promising diagnostic and therapeutic tools for a variety of diseases. The characterization of EVs requires a series of orthogonal techniques that are overall time- and material-consuming. Here, a microfluidic device is presented that exploits the combination of diffusion sizing and multiwavelength fluorescence detection to simultaneously provide information on EV size, concentration, and composition. The latter is achieved with the nonspecific staining of lipids and proteins combined with the specific staining of EV markers such as EV-associated tetraspanins via antibodies. The device can be operated as a single-step immunoassay thanks to the integrated separation and quantification of free and EV-bound fluorophores. This microfluidic technique is capable of detecting and quantifying components associated to EV subtypes and impurities and thus to measure EV purity in a time scale of minutes, requiring less than 5 µL of sample and minimal sample handling before the analysis. Moreover, the analysis is performed directly in solution without immobilization steps. Therefore, this method can accelerate screening of EV samples and aid the evaluation of sample reproducibility, representing an important complementary tool to the current array of biophysical methods for EV characterization, particularly valuable for instance for bioprocess development.

A Molecular Logic Gate Enables Single-Molecule Imaging and Tracking of Lipids in Intracellular Domains.

Photoactivatable dyes enable single-molecule imaging and tracking in biology. Despite progress in the development of new fluorophores and labeling strategies, many intracellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid domains, e.g., membranes and droplets, remain difficult to image with nanometric resolution. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid domains, most notably droplets, with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment, and a competent nucleophile to produce a fluorescent product. The combination of these inputs results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolution beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipid trafficking between droplets and the endoplasmic reticulum.