Advancing multiscale structural mapping of the brain through fluorescence imaging and analysis across length scales

Abstract

Brain function emerges from hierarchical neuronal structure that spans orders of magnitude in length scale, from the nanometre-scale organization of synaptic proteins to the macroscopic wiring of neuronal circuits. Because the synaptic electrochemical signal transmission that drives brain function ultimately relies on the organization of neuronal circuits, understanding brain function requires an understanding of the principles that determine hierarchical neuronal structure in living or intact organisms. Recent advances in fluorescence imaging now enable quantitative characterization of neuronal structure across length scales, ranging from single-molecule localization using super-resolution imaging to whole-brain imaging using light-sheet microscopy on cleared samples. These tools, together with correlative electron microscopy and magnetic resonance imaging at the nanoscopic and macroscopic scales, respectively, now facilitate our ability to probe brain structure across its full range of length scales with cellular and molecular specificity. As these imaging datasets become increasingly accessible to researchers, novel statistical and computational frameworks will play an increasing role in efforts to relate hierarchical brain structure to its function. In this perspective, we discuss several prominent experimental advances that are ushering in a new era of quantitative fluorescence-based imaging in neuroscience along with novel computational and statistical strategies that are helping to distil our understanding of complex brain structure.

Keywords: brain structure; fluorescence imaging; multiscale analysis and modelling.

Figure 1.

Multiscale modelling of brain imaging data. (a) Co-localization of GluA1 and PSD95 in dissociated rat hippocampal neurons (left), Manders' co-localization of GluA1 and PSD95 during elevated glycine exposure relative to control conditions (centre) [16] and equations for Manders' coefficents (right). (b) Morphological changes induced by the neuroligin-3 mutation R451C in mice (left) [17], dendritic size and complexity as analysed with the Sholl method in mouse SHANK3 knockout tissue (centre) [18] and Sholl equations (right). (c) EM data (left) and circuit reconstruction (centre) in the inner plexiform layer of the mouse retina [19]. The relationship between node degree and the number of edges in a complete directed graph (right). (d) Inter-regional connectivity data (left) is used to create a node degree distribution (centre) from tracer injections at hundreds of unique sites [20]. Node degree distributions that match a power law distribution are indicative of a scale-free network structure (right). (e) Modularity and hierarchy of network maps as assessed by structural MRI data [21]. (Online version in colour.)

Figure 2.

Super-resolution imaging of neuronal structure. (a) (Left) PALM/STORM of mEos2-gephyrin (red) and Alexa 647-labelled endogenous GlyRa1 (cyan) in fixed spinal cord neurons shows the correspondence between the GlyR and gephyrin distributions at inhibitory synapses. Also note the co-localization (within less than 50 nm) of GlyRs and gephyrin nanoclusters (arrowhead). Scale: box width of 1.25 μm. (Right) Quantification of the number of mRFP-gephyrin molecules in cortex (black) and in spinal cord inhibitory synapses that are negative (blue) or positive (red) for endogenous GlyRa1. (b) A composite plot of the axial positions of synaptic proteins imaged using three-colour STORM. For each protein, the coloured dot specifies the mean axial position, the two vertical lines represent the associated SEM and the half-length of the horizontal bar denotes the s.d., derived from multiple synapses. (c) Molecular multiplexing with correlative EM with AT. A SEM field is shown in greyscale, with immunofluorescence for VGluT1 (light blue), glutamine synthetase (orange), synapsin-1 (green) and PSD-95 (red). (d) (Top) Single-molecule localization of PSD-95-Eos2. Individual molecules were colour coded according to their local density. (Middle) Homogeneous distribution generated by randomly sampling equal numbers of localizations is observed. Scale bar, 100 nm. (Bottom) Mean pair-correlation function of the PSD in the measured particle locations (blue) and for the simulated locations (red). Shaded areas represent 99% CIs calculated from the randomized ensembles, showing significant departures from homogeneity. (Online version in colour.)

Figure 3.

Three-dimensional intact imaging of brains and organoids. (a) The Brainbow construct results in combinatorial and unique XFP expressions; oculomotor axons of Thy1-Brainbow-1.0 line H and dentate gyrus of Thy1-Brainbow-1.0 line L [64]. (b) Three-dimensional reconstructed images of mouse brains expressing various fluorescent proteins were acquired with light-sheet microscopy. Ventral-to-dorsal images of three different transgenic mouse strains [65]. (c) Pre-frontal cortex of PACT-cleared adult mouse brain sections stained with antibodies against GFAP (glial fibrillary acidic protein), murine immunoglobulin G (IgG) and ionized calcium-binding adaptor molecule 1 (Iba1) [7]. (Online version in colour.)

Figure 4.

Tissue and disease modelling with cerebral organoids. (a) Brain region identity is captured using human cerebral organoids; fluorescence imaging for PAX6 (forebrain marker), KROX20 (hindbrain marker) and PAX2 (hindbrain marker); hippocampal markers NRP2, FZD9, PROX1 [80]. (b) Control-derived spheroids form thick fluid-filled cortical tissues. Microcephaly patient-derived tissues (line 14B) display neuroepithelium and outgrowth of neuronal morphology as compared to control [80]. (c) Modelling of microcephaly through cerebral organoids; day 22 staining indicates higher number of neurons (TUJ1, arrows) in patient-derived tissue [80]. (d) Comparison between six-week differentiated control (ReN-G) and familial Alzheimer's disease (FAD) ReN cells (ReN-GA, ReN-mGAP). Detection of amyloid plaques in ReN-mGAP cells with Amylo-Glo (green—GFP, red—3D6 anti-amyloid-β, blue—Amylo-Glo, arrows—Amylo-Glo-positive aggregates) [81]. (Online version in colour.)