Fig. 3 |. The SHIELD–MAP and AAV-based labelling system.

SHIELD (system-wide control of interaction time and kinetics of chemicals) combined with MAP (magnified analysis of proteome) allows integrated circuit mapping at single-cell resolution. a | The SHIELD–MAP pipeline. SHIELD allows fully integrated multiscale imaging of fluorescent protein-labelled circuits, mRNAs and proteins within the same mouse brain by simultaneously protecting the molecular and structural information within cleared tissues. b | The image shows a 3D rendering of fluorescent protein-labelled neuronal circuitry of parvalbumin (PV)-positive neurons in the globus pallidus externa (GPe) with an overlaid axon trace of a single labelled neuron. The inset shows example images from multi round staining and multiscale imaging. Scale bar 50 μm for the insets. c | Reconstructed axon arborization of the neuron and its downstream targets. Each circle represents a neuron. The number of putative axosomatic boutons is marked inside each circle. The colour of each circle provides molecular details for each neuron. d | Reconstructed putative axosomatic connectivity for ‘cell D’ (the cell highlighted in orange in part c). Ramified axons (grey) and enhanced green fluorescent protein (eCFP)-positive presynaptic boutons (blue) are segmented⁷¹. Scale bars 20 μm. e | Viral-assisted spectral tracing (VAST) can be used to label and visualize the 3D morphology and connectivity of cells in thick, cleared tissue blocks. Here, the schematic shows the two-component VAST labelling system^30,80,122. A high dose of a three-vector cocktail (individually expressing a red, green or blue fluorescent protein) is coadministered along with a variable dose of an inducer vector that is required to turn on expression of the three proteins. To label specific cell populations, the expression of the fluorescent proteins can also be made Cre dependent. f | Projection image of the olfactory bulb where mitral cells are labelled by VAST (left). Sparse labelling of these cells allows tracing of their dendritic arbors (middle). An overlay of the traces and the projection image is shown in right-hand image. AAV, adeno-associated virus; CR, calretinin; FISH, fluorescence in situ hybridization; hSyn1, human synapsin 1 gene promoter; IHC, immunohistochemistry; GPi, globus pallidus interna; nRT, nucleus reticularis thalami; SNr, substantia nigra pars reticulata; STN,subthalamic nucleus; WPRE, Woodchuck hepatitis post-transcriptional regulatory element. Parts a–d are adapted from REF.⁷¹, Springer Nature Limited. Parts e and f are adapted from REF.⁸⁰, Springer Nature Limited.