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INTRODUCTION Neural circuits across the brain are composed of structures spanning seven orders of magnitude in size that are assembled from thousands of distinct protein types. Electron microscopy has imaged densely labeled brain tissue at nanometer-level resolution over near-millimeter-level dimensions but lacks the contrast to distinguish specific proteins and the speed to readily image multiple specimens. Conversely, confocal fluorescence microscopy offers molecular contrast but has insufficient resolution for dense neural tracing or the precise localization of specific molecular players within submicrometer-sized structures. Last, superresolution fluorescence microscopy bleaches fluorophores too quickly for large-volume imaging and also lacks the speed for effective brain-wide or cortex-wide imaging of multiple specimens. RATIONALE We combined two imaging technologies to address these issues. Expansion microscopy (ExM) creates an expanded, optically clear phantom of a fluorescent specimen that retains its original relative distribution of fluorescent tags. Lattice light-sheet microscopy (LLSM) then images this phantom in three dimensions with minimal photobleaching at speeds sufficient to image the entire Drosophila brain or across the width of the mouse cortex in ∼2 to 3 days, with multiple markers at an effective resolution of ∼60 by 60 by 90 nm for 4× expansion. RESULTS We applied expansion/LLSM (ExLLSM) to study a variety of subcellular structures in the brain. In the mouse cortex, we quantified the volume of organelles, measured morphological parameters of ~1500 dendritic spines, determined the variation of distances between pre- and postsynaptic proteins, observed large differences in postsynaptic expression at adjacent pyramidal neurons, and studied both the azimuthal asymmetry and layer-specific longitudinal variation of axonal myelination. In Drosophila , we traced the axonal branches of olfactory projection neurons across one hemisphere and studied the stereotypy of their boutons at the calyx and lateral horn across five animals. We also imaged all dopaminergic neurons (DANs) across the brain of another specimen, visualized DAN morphologies in all major brain regions, and traced a cluster of eight DANs to their termini to determine their respective cell types. In the same specimen, we also determined the number of presynaptic active zones (AZs) across the brain and the local density of all AZs and DAN-associated AZs in each brain region. CONCLUSION With its high speed, nanometric resolution, and ability to leverage genetically targeted, cell type–specific, and protein-specific fluorescence labeling, ExLLSM fills a valuable niche between the high throughput of conventional optical pipelines of neural anatomy and the ultrahigh resolution of corresponding EM pipelines. Assuming the development of fully validated, brain-wide isotropic expansion at 10× or beyond and sufficiently dense labeling, ExLLSM may enable brainwide comparisons of even densely innervated neural circuits across multiple specimens with protein-specific contrast at 25-nm resolution or better.

The sequence of the Drosophila melanogaster genome presented in this issue of Science is the latest milestone in nine decades of research on this organism. Genetic and physical mapping, whole-genome mutational screens, and functional alteration of the genome by gene transfer were pioneered in metazoans with the use of this small fruit fly. Here we look at some of the instances in which work on Drosophila has led to major conceptual or technical breakthroughs in our understanding of animal genomes.

Recombinant DNA carrying the 3-kilobase transposable element was injected into Drosophila embryos of a strain that lacked such elements. Under optimum conditions, half of the surviving embryos showed evidence of P element-induced mutations in a fraction of their progeny. Direct analysis of the DNA of strains derived from such flies showed them to contain from one to five intact 3-kilobase P elements located at a wide variety of chromosomal sites. DNA sequences located outside the P element on the injected DNA were not transferred. Thus P elements can efficiently and selectively transpose from extrachromosomal DNA to the DNA of germ line chromosomes in Drosophila embryos. These observations provide the basis for efficient DNA-mediated gene transfer in Drosophila.

Phototransduction is the process by which light-stimulated photoreceptor cells of the visual system send electrical signals to the nervous system. Many of the steps that follow the initial event in phototransduction, absorption of light by rhodopsin, are ill-defined. The fruitfly, Drosophila melanogaster, provides a means to dissect phototransduction genetically. Mutations such as transient receptor potential (trp) affect intermediate steps in phototransduction. In order to facilitate molecular studies of phototransduction, the trp gene was isolated and its identity was confirmed by complementing the mutant trpCM allele of the trp gene by P-element mediated germline transformation of a 7.1-kilobase DNA fragment. Expression of the trp gene begins late in pupal development and appears to be limited to the eyes and ocelli.

The fruit fly Drosophila melanogaster has been used as an experimental organism in studies of genetics since the early 1900s. It is now widely used not only in classical and molecular genetics but also, with many new biochemical, cell biological, and physiological techniques, to research problems requiring a multidisciplinary approach, such as those of developmental biology and neurobiology.