The development of single-molecule localization microscopy (SMLM) has sparked a revolution in biological imaging, allowing 'super-resolution' fluorescence microscopy below the diffraction limit of light. The last decade has seen an explosion in not only optical hardware for SMLM but also the development or repurposing of fluorescent proteins and small-molecule fluorescent probes for this technique. In this review, written by chemists for chemists, we detail the history of single-molecule localization microscopy and collate the collection of probes with demonstrated utility in SMLM. We hope it will serve as a primer for probe choice in localization microscopy as well as an inspiration for the development of new fluorophores that enable imaging of biological samples with exquisite detail.

Brain function is mediated by neural circuit connectivity, and elucidating the role of connections is aided by techniques to block their output. We developed cell-type-selective, reversible synaptic inhibition tools for mammalian neural circuits by leveraging G protein signaling pathways to suppress synaptic vesicle release. Here, we find that the pharmacologically selective designer Gi-protein-coupled receptor hM4D is a presynaptic silencer in the presence of its cognate ligand clozapine-N-oxide (CNO). Activation of hM4D signaling sharply reduced synaptic release probability and synaptic current amplitude. To demonstrate the utility of this tool for neural circuit perturbations, we developed an axon-selective hM4D-neurexin variant and used spatially targeted intracranial CNO injections to localize circuit connections from the hypothalamus to the midbrain responsible for feeding behavior. This synaptic silencing approach is broadly applicable for cell-type-specific and axon projection-selective functional analysis of diverse neural circuits.

Chemogenetic technologies enable selective pharmacological control of specific cell populations. An increasing number of approaches have been developed that modulate different signaling pathways. Selective pharmacological control over G protein-coupled receptor signaling, ion channel conductances, protein association, protein stability, and small molecule targeting allows modulation of cellular processes in distinct cell types. Here, we review these chemogenetic technologies and instances of their applications in complex tissues in vivo and ex vivo.

Elucidating the roles of neuronal cell types for physiology and behavior is essential for understanding brain functions. Perturbation of neuron electrical activity can be used to probe the causal relationship between neuronal cell types and behavior. New genetically encoded neuron perturbation tools have been developed for remotely controlling neuron function using small molecules that activate engineered receptors that can be targeted to cell types using genetic methods. Here we describe recent progress for approaches using genetically engineered receptors that selectively interact with small molecules. Called "chemogenetics," receptors with diverse cellular functions have been developed that facilitate the selective pharmacological control over a diverse range of cell-signaling processes, including electrical activity, for molecularly defined cell types. These tools have revealed remarkably specific behavioral physiological influences for molecularly defined cell types that are often intermingled with populations having different or even opposite functions.

Chromatin immunoprecipitation (ChIP) is a technique that reveals in vivo location of a protein bound to DNA. ChIP coupled with DNA microarrays (ChIP-chip) or next-generation sequencing (ChIP-seq) allows for identification of binding sites of transcription factors on a global scale. Here we describe a protocol for ChIP to identify binding of the Ultrabithorax (Ubx) Hox transcription factors from imaginal discs of Drosophila larvae. The protocol can be extended to other model organisms and transcription factors.

Plastid isoprenoid-derived carotenoids serve essential roles in chloroplast development and photosynthesis. Although nearly all enzymes that participate in the biosynthesis of carotenoids in plants have been identified, the complement of auxiliary proteins that regulate synthesis, transport, sequestration, and degradation of these molecules and their isoprenoid precursors have not been fully described. To identify such proteins that are necessary for the optimal functioning of oxygenic photosynthesis, we screened a large collection of nonphotosynthetic (acetate-requiring) DNA insertional mutants of and isolated The mutant is extremely light-sensitive and susceptible to photoinhibition and photobleaching. The gene encodes a CRAL-TRIO hydrophobic ligand-binding (Sec14) domain protein. Proteins containing this domain are limited to eukaryotes, but some may have been retargeted to function in organelles of endosymbiotic origin. The mutant showed decreased accumulation of plastidial isoprenoid-derived pigments, especially carotenoids, and whole-cell focused ion-beam scanning-electron microscopy revealed a deficiency of carotenoid-rich chloroplast structures (e.g., eyespot and plastoglobules). The low carotenoid content resulted from impaired biosynthesis at a step prior to phytoene, the committed precursor to carotenoids. The CPSFL1 protein bound phytoene and β-carotene when expressed in and phosphatidic acid in vitro. We suggest that CPSFL1 is involved in the regulation of phytoene synthesis and carotenoid transport and thereby modulates carotenoid accumulation in the chloroplast.

Cells require a constant supply of fatty acids to survive and proliferate. Fatty acids incorporate into membrane and storage glycerolipids through a series of endoplasmic reticulum (ER) enzymes, but how these enzymes are regulated is not well understood. Here, using a combination of CRISPR-based genetic screens and unbiased lipidomics, we identified calcineurin B homologous protein 1 (CHP1) as a major regulator of ER glycerolipid synthesis. Loss of CHP1 severely reduces fatty acid incorporation and storage in mammalian cells and invertebrates. Mechanistically, CHP1 binds and activates GPAT4, which catalyzes the initial rate-limiting step in glycerolipid synthesis. GPAT4 activity requires CHP1 to be N-myristoylated, forming a key molecular interface between the two proteins. Interestingly, upon CHP1 loss, the peroxisomal enzyme, GNPAT, partially compensates for the loss of ER lipid synthesis, enabling cell proliferation. Thus, our work identifies a conserved regulator of glycerolipid metabolism and reveals plasticity in lipid synthesis of proliferating cells.

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

Modern microscopy methods incorporate computational modeling of optical systems as an integral part of the imaging process, either to solve inverse problems or enable optimization of the optical system design. These methods often depend on differentiable simulations of optical systems, yet no standardized framework exists—forcing computational optics researchers to repeatedly and independently implement simulations that are prone to errors, difficult to reuse in other applications, and often computationally suboptimal. These common problems limit the potential impact of computational optics as a field. We present Chromatix: an open-source, GPU-accelerated differentiable wave optics library. Chromatix builds on JAX to enable fast simulation of diverse optical systems and inverse problem solving, scaling these simulations from single-CPU laptops to multi-GPU servers. The library implements various optical elements (e.g., lenses, polarizers and spatial light modulators) and multiple light propagation models (e.g., Fresnel approximation, angular spectrum and off-axis propagation) that can be flexibly combined to model various computational optics applications such as snapshot microscopy, holography, and phase retrieval of multiple scattering samples. These simulations can be automatically parallelized to scale across multiple GPUs with a single-line change to the modeling code, enabling simulation and optimization of previously impractical optical system designs. We demonstrate Chromatix’s capacity to substantially accelerate optics simulation and optimization on existing methods in computational optics, speeding up optical simulation and optimization from 2-6× on a single GPU to up to 22× on 8 GPUs (depending on the particular system being modeled) compared to the original implementations. Chromatix establishes a standard for wave optics simulations, democratizing access to and expanding the design space of computational optics.i

The emergence of new and increasingly sophisticated behaviors after birth is accompanied by dramatic increase of newly established synaptic connections in the nervous system. Little is known, however, of how nascent connections are organized to support such new behaviors alongside existing ones. To understand this, in the larval zebrafish we examined the development of spinal pathways from hindbrain V2a neurons and the role of these pathways in the development of locomotion. We found that new projections are continually layered laterally to existing neuropil, and give rise to distinct pathways that function in parallel to existing pathways. Across these chronologically layered pathways, the connectivity patterns and biophysical properties vary systematically to support a behavioral repertoire with a wide range of kinematics and dynamics. Such layering of new parallel circuits equipped with systematically changing properties may be central to the postnatal diversification and increasing sophistication of an animal's behavioral repertoire.