Whole-brain serial-section electron microscopy in larval zebrafish

Abstract

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits(1). However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons(2), some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques(1), but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure-function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource

Correlating 3D light to 3D electron microscopy for systems biology

“…For example, in the brain, while the projectome may turn out to be comparable between individuals, connectomes are likely to vary at the level of individual synapses. Importantly, the work of Hildebrand et al [35] demonstrates the feasibility of taking an individual animal through a full workflow including functional imaging to serial section SEM. Further optimization of a pipeline for 3D CLEM and integrated workflows will push correlative measurements in biological systems to even richer meta datasets.…”

“…Automation of serial section collection onto kapton tape, which is then placed onto wafers and imaged in an SEM, has delivered large volume reconstructions of mouse neocortex [34] and a whole zebrafish brain [35]. Maintaining the library of sections on a wafer is advantageous as it allows reinspection of the sections and thus sequential steps of low-magnification pre-scanning with highermagnification re-scanning of targeted sections or areas.…”

“…In LM, remarkably, registration of volumetrically morphed confocal z-stacks has allowed mapping of anatomy of populations of age-matched individual zebrafish larvae with near cellular resolution [66]. Registration with EM volume maps [35] may raise the possibility of comparisons of macroscopic distributions of ultrastructural features between individual animals. However, certain classes of problems are inherently difficult to address at the population level.…”

“…Data collection can be optimized via smart tracking algorithms that detect features, landmarks, or probes identified by LM or X-ray microscopy to drive data collection from regions of interest for EM. Array tomography approaches have already used low-resolution EM pre-scanning to determine regions for higher resolution re-scanning [34,35]. With its large specificity and ease of recognition, fluorescence seems optimally suited for fast identification of target areas using an integrated microscope.…”

“…However, after sectioning, the (two-dimensional) coordinate transformation imposed by the sectioning may be different for each section. Thus, while serial sectioning involves careful non-linear section-to-section registration [35], blockface techniques deliver aligned high-resolution image stacks through large volumes. Indeed, SBEM and FIB-SEM have both already been used to image volumes of brain and other tissues [38][39][40][41].…”