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.

Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) that are functionally brighter; detect submicromolar to millimolar amounts of glutamate; and have blue, cyan, green, or yellow emission profiles. These variants could be imaged in vivo in cases where original iGluSnFR was too dim, resolved glutamate transients in dendritic spines and axonal boutons, and allowed imaging at kilohertz rates.

Dopamine is a key chemical neuromodulator that plays vital roles in various brain functions. Traditionally, neuromodulators like dopamine are believed to be released in a diffuse manner and are not commonly associated with synaptic structures where pre- and postsynaptic processes are closely aligned. Our findings challenge this conventional view. Using single-bouton optical measurements of dopamine release, we discovered that dopamine is predominantly released from varicosities that are juxtaposed against the processes of their target neurons. Dopamine axons specifically target neurons expressing dopamine receptors, forming synapses to release dopamine. Interestingly, varicosities that were not directly apposed to dopamine receptor-expressing processes or associated with neurons lacking dopamine receptors did not release dopamine, regardless of their vesicle content. The ultrastructure of dopamine release sites share common features of classical synapses. We further show that the dopamine released at these contact sites induces a precise, dopamine-gated biochemical response in the target processes. Our results indicate that dopamine release sites share key characteristics of conventional synapses that enable relatively precise and efficient neuromodulation of their targets.

The development of genetically encoded self-labeling protein tags such as the HaloTag and SNAP-tag has expanded the utility of chemical dyes in microscopy. Intracellular labeling using these systems requires small, cell-permeable dyes with high brightness and photostability. We recently discovered a general method to improve the properties of classic fluorophores by replacing N,N-dimethylamino groups with four-membered azetidine rings to create the "Janelia Fluor" dyes. Here, we describe the synthesis of the HaloTag and SNAP-tag ligands of Janelia Fluor 549 and Janelia Fluor 646 as well as standard labeling protocols for use in ensemble and single-molecule cellular imaging.

Chemigenetic tags are versatile labels for fluorescence microscopy that combine some of the advantages of genetically encoded tags with small molecule fluorophores. The Fluorescence Activating and absorbance Shifting Tags (FASTs) bind a series of highly fluorogenic and cell-permeable chromophores. Furthermore, FASTs can be used in complementation-based systems for detecting or inducing protein-protein interactions, depending on the exact FAST protein variant chosen. In this study, we systematically explore substitution patterns on FAST fluorogens and generate a series of fluorogens that bind to FAST variants, thereby activating their fluorescence. This effort led to the discovery of a novel fluorogen with superior properties, as well as a fluorogen that transforms splitFAST systems into a fluorogenic dimerizer, eliminating the need for additional protein engineering.

Glutamate is the principal excitatory neurotransmitter, and occasionally subserves inhibitory roles, in the vertebrate nervous system. Glutamatergic synapses are dense in the vertebrate brain, at \textasciitilde1/μm3. Glutamate is released from and onto diverse components of the nervous system, including neurons, glia, and other cells. Methods for glutamate detection are critically important for understanding the function of synapses and neural circuits in normal physiology, development, and disease. Here we describe the development, optimization, and deployment of genetically encoded fluorescent glutamate indicators. We review the theoretical considerations governing glutamate sensor properties from first principles of synapse biology, microscopy, and protein structure-function relationships. We provide case studies of the state-of-the-art iGluSnFR glutamate sensor, encompassing design and optimization, mechanism of action, in vivo imaging, data analysis, and future directions. We include detailed protocols for iGluSnFR imaging in common preparations (bacteria, cell culture, and brain slices) and model organisms (worm, fly, fish, rodent).