Combining the molecular specificity of fluorescent probes with three-dimensional imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a 4Pi single-molecule switching super-resolution microscope that enables ratiometric multicolor imaging of mammalian cells at 5-10-nm localization precision in three dimensions using 'salvaged fluorescence'. Imaging two or three fluorophores simultaneously, we show fluorescence images that resolve the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy. The salvaged fluorescence approach is equally applicable in most single-objective microscopes.

Calcium carbonate platforms produced by reef-building stony corals over geologic time are pervasive features around the world [1]; however, the mechanism by which these organisms produce the mineral is poorly understood (see review by [2]). It is generally assumed that stony corals precipitate calcium carbonate extracellularly as aragonite in a calcifying medium between the calicoblastic ectoderm and pre-existing skeleton, separated from the overlying seawater [2]. The calicoblastic ectoderm produces extracellular matrix (ECM) proteins, secreted to the calcifying medium [3-6], which appear to provide the nucleation, alteration, elongation, and inhibition mechanisms of the biomineral [7] and remain occluded and preserved in the skeleton [8-10]. Here we show in cell cultures of the stony coral Stylophora pistillata that calcium is concentrated in intracellular pockets that are subsequently exported from the cell where a nucleation process leads to the formation of extracellular aragonite crystals. Analysis of the growing crystals by lattice light-sheet microscopy suggests that the crystals elongate from the cells' surfaces outward.

Optical nanoscopy of intact biological specimens has been transformed by recent advancements in hydrogel-based tissue clearing and expansion, enabling the imaging of cellular and subcellular structures with molecular contrast. However, existing high-resolution fluorescence microscopes have limited imaging depth, which prevents the study of whole-mount specimens without physical sectioning. To address this challenge, we developed “photochemical sectioning,” a spatially precise, light-based sample sectioning process. By combining photochemical sectioning with volumetric lattice light-sheet imaging and petabyte-scale computation, we imaged and reconstructed axons and myelination sheaths across entire mouse olfactory bulbs at nanoscale resolution. An olfactory-bulb-wide analysis of myelinated and unmyelinated axons revealed distinctive patterns of axon degeneration and de-/dysmyelination in the neurodegenerative mouse, highlighting the potential for peta- to exabyte-scale super-resolution studies using this approach.

How neuromodulatory transmitters diffuse into the extracellular space remains an unsolved fundamental biological question, despite wide acceptance of the volume transmission model. Here, we report development of a method combining genetically encoded fluorescent sensors with high-resolution imaging and analysis algorithms which permits the first direct visualization of neuromodulatory transmitter diffusion at various neuronal and non-neuronal cells. Our analysis reveals that acetylcholine and monoamines diffuse at individual release sites with a spread length constant of ∼0.75 μm. These transmitters employ varied numbers of release sites, and when spatially close-packed release sites coactivate they can spillover into larger subcellular areas. Our data indicate spatially restricted (i.e., nonvolume) neuromodulatory transmission to be a prominent intercellular communication mode, reshaping current thinking of control and precision of neuromodulation crucial for understanding behaviors and diseases.

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function.

Mammalian mtDNA has been found here to harbor RNA-DNA hybrids at a variety of locations throughout the genome. The R-loop, previously characterized in vitro at the leading strand replication origin (OH), is isolated as a native RNA-DNA hybrid copurifying with mtDNA. Surprisingly, other mitochondrial transcripts also form stable partial R-loops. These are abundant and affect mtDNA conformation. Current models regarding the mechanism of mammalian mtDNA replication have been expanded by recent data and discordant hypotheses. The presence of stable, nonreplicative, and partially hybridized RNA on the mtDNA template is significant for the reevaluation of replication models based on two-dimensional agarose gel analyses. In addition, the close association of a subpopulation of mtRNA with the DNA template has further implications regarding the structure, maintenance, and expression of the mitochondrial genome. These results demonstrate that variously processed and targeted mtRNAs within mammalian mitochondria likely have multiple functions in addition to their conventional roles.

Animal species display enormous variation for innate behaviours, but little is known about how this diversity arose. Here, using an unbiased genetic approach, we map a courtship song difference between wild isolates of Drosophila simulans and Drosophila mauritiana to a 966 base pair region within the slowpoke (slo) locus, which encodes a calcium-activated potassium channel. Using the reciprocal hemizygosity test, we confirm that slo is the causal locus and resolve the causal mutation to the evolutionarily recent insertion of a retroelement in a slo intron within D. simulans. Targeted deletion of this retroelement reverts the song phenotype and alters slo splicing. Like many ion channel genes, slo is expressed widely in the nervous system and influences a variety of behaviours; slo-null males sing little song with severely disrupted features. By contrast, the natural variant of slo alters a specific component of courtship song, illustrating that regulatory evolution of a highly pleiotropic ion channel gene can cause modular changes in behaviour.

In animals, scaling relationships between appendages and body size exhibit high interspecific variation but low intraspecific variation. This pattern could result from natural selection for specific allometries or from developmental constraints on patterns of differential growth. We performed artificial selection on the allometry between forewing area and body size in a butterfly to test for developmental constraints, and then used the resultant increased range of phenotypic variation to quantify natural selection on the scaling relationship. Our results show that the short-term evolution of allometries is not limited by developmental constraints. Instead, scaling relationships are shaped by strong natural selection.