The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone–zwitterion equilibrium constant (KL–Z) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure–activity relationships that govern KL–Z. We discovered that the auxochrome substituent strongly affects the lactone–zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Förster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure–activity relationships that will guide the design of future probes.

The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of membrane and secretory proteins. Specific ER subdomains are thought to be spatially organized to enable protein synthesis activity, but precisely where these domains are localized is unclear, especially relative to the plethora of organelle interactions taking place on the ER. Here, we use single-molecule tracking of ribosomes and mRNA in combination with simultaneous imaging of ER to assess the sites of membrane protein synthesis on the ER. We found that ribosomes were widely distributed throughout different ER morphologies, but the synthesis of membrane proteins (including Type I, II, and multi-spanning) and an ER luminal protein (Calreticulin) occurred primarily at three-way junctions. Lunapark played a key role in stabilizing transmembrane protein mRNA at three-way junctions. We additionally found that translating mRNAs coding for transmembrane proteins are in the vicinity of lysosomes and translate through a cap-independent but eIF2-dependent mechanism. These results support the idea that discrete ER subdomains co-exist with lysosomes to support specific types of protein synthesis activities, with ER-lysosome interactions playing an important role in the translation of secretome mRNAs.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.

Light-mediated chemical reactions are powerful methods for manipulating and interrogating biological systems. Photosensitizers, compounds that generate reactive oxygen species upon excitation with light, can be utilized for numerous biological experiments, but the repertoire of bioavailable photosensitizers is limited. Here, we describe the synthesis, characterization, and utility of two photosensitizers based upon the widely used rhodamine scaffold and demonstrate their efficacy for chromophore-assisted light inactivation, cell ablation in culture and in vivo, and photopolymerization of diaminobenzidine for electron microscopy. These chemical tools will facilitate a broad range of applications spanning from targeted destruction of proteins to high-resolution imaging.

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant () is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.

The presumptive altered dynamics of transient molecular interactions in vivo contributing to neurodegenerative diseases have remained elusive. Here, using single-molecule localization microscopy, we show that disease-inducing Huntingtin (mHtt) protein fragments display three distinct dynamic states in living cells - 1) fast diffusion, 2) dynamic clustering and 3) stable aggregation. Large, stable aggregates of mHtt exclude chromatin and form 'sticky' decoy traps that impede target search processes of key regulators involved in neurological disorders. Functional domain mapping based on super-resolution imaging reveals an unexpected role of aromatic amino acids in promoting protein-mHtt aggregate interactions. Genome-wide expression analysis and numerical simulation experiments suggest mHtt aggregates reduce transcription factor target site sampling frequency and impair critical gene expression programs in striatal neurons. Together, our results provide insights into how mHtt dynamically forms aggregates and disrupts the finely-balanced gene control mechanisms in neuronal cells.