Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation.

The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins.

Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins.

Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out.

Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen activating proteins.

Agonist-promoted G-protein coupled receptor (GPCR) endocytosis and recycling plays an important role in many signaling events in the cell. However, the approaches that allow fast and quantitative analysis of such processes still remain limited. Here we report an improved labeling approach based on the genetic fusion of a fluorogen activating protein (FAP) to a GPCR and binding of a sulfonated analog of the malachite green (MG) fluorogen to rapidly and selectively label cell surface receptors.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces.

We report that a symmetric small-molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* fluorogen activating protein is a VL domain that binds malachite green (MG) dye to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains.

Nanoparticle transport from mouse vagina to adjacent lymph nodes.

To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes.

Imaging vasculature and lymphatic flow in mice using quantum dots.

Quantum dots are ideal probes for fluorescent imaging of vascular and lymphatic tissues. On injection into appropriate sites, red- and near-infrared-emitting quantum dots provide excellent definition of vasculature, lymphoid organs, and lymph nodes draining both normal tissues and tumors. We detail methods for use with commercially available quantum dots and discuss common difficulties.

Long-term persistence and spectral blue shifting of quantum dots in vivo.

Quantum dots are a powerful fluorophore family with desirable attributes for fluorescence imaging. They have been used in several animal models with direct clinical relevance, including sentinel lymph node mapping, tracing vasculature and lymphatics, and targeting specific lesions for diagnosis and removal. (1-12) Despite significant interest for use in translational applications, little is known about the persistence and long-term fate of quantum dots in vivo.

Cholera toxin B conjugated quantum dots for live cell labeling.

Cholera toxin subunit B (CTB)--quantum dot conjugates were developed for labeling mammalian cells. The conjugates were internalized by all tested cell lines into small vesicles dispersed throughout the cytoplasm, while commercially available polyarginine conjugates rapidly accumulated in large perinuclear endosomes. Although a large proportion of CTB conjugates eventually also accumulated in perinuclear endosomes, this accumulation required several days, and even then many CTB conjugated quantum dots remained in small vesicles dispersed throughout the cytoplasm.

Sentinel lymph node imaging using quantum dots in mouse tumor models.

We demonstrate that quantum dots injected into two model tumors rapidly migrate to sentinel lymph nodes. PEG-coated quantum dots having terminal carboxyl, amino, or methoxyl groups all migrated from the tumor to surrounding lymph nodes similarly. Passage from the tumor through lymphatics to adjacent nodes could be visualized dynamically through the skin; at least two nodes could usually be defined.