Serial two-photon tomography for automated ex vivo mouse brain imaging

Abstract

Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.

Figure 1

STP tomography. (a) Schema of the method. Computer-controlled XYZ stages move the brain under the objective of a two-photon microscope, so that the top view is imaged as a mosaic. The stages also deliver the brain to a built-in vibrating blade microtome for sectioning (Supplementary Video 1). (b) Coronal (top), horizontal (mid) and sagittal (bottom) views of a GFPM dataset of 260 coronal sections (Supplementary Fig. 1) after 3D reconstruction. (c) 3D view of a coronal section of the GFPM brain imaged with a 20x objective at 0.5 μm XY sampling. Lower left: position of the coronal plane in the imaged mouse brain (approximately −2.5 mm from Bregma). (d-e) Enlarged views from regions (i) and (ii) in panel (c), respectively, demonstrating visualization of dendritic spines (d) and fine axon fibers (e); scale bar = 25 μm (d-e) and 5 μm (insert in d). See Supplementary Figs. 2 and 3 for a comparison of different imaging conditions.

Figure 2

Retrograde tracing by CTB-Alexa-488. (a) 3D view of a coronal section comprising the injection site (i) and several retrogradely labeled regions (ii-iv). Lower left: position of the section in the whole brain (approximately −1.15 mm from Bregma). (b) Coronal (top), sagittal (bottom), and horizontal views of the injection site. (c) Cortical regions marked up in (a), comprising: (i) the injection site in the barrel field of the primary somatosensory cortex (S1BF), (ii) ipsilateral secondary somatosensory cortex (S2), (iii) granular insular cortex (GI), and (iv) contralateral S1BF. The panels (ii-iv) are shown with enlarged regions from supragranular and infragranular cortical layers comprising CTB labeled cells. The scale bar is 250 μm in panel (i) and 50 μm in the enlarged view of panel (ii).

Figure 3

Anterograde tracing by AAV-GFP and brain warping. (a) 3D view of a coronal section comprising the injection site (i) and several anterogradely labeled regions (ii-v). Lower left: position of the section in the whole brain (approximately −1.9 mm from Bregma). (b) Coronal (top) and sagittal (bottom) views of the injection site. (c) The injection site (S1BF). (d) Brain regions (ii-v) marked up in (a), comprising: (ii) ipsilateral caudoputamen (CP); (iii) axon fibers in the internal capsule (ic) (iv) ventral posteromedial thalamus (VPM) and posterior thalamus (PO), and (v) contralateral barrel cortex (S1BF). The enlarged views show inverted grayscale images for better visualization of axon fibers and varicosities. The scale bar in (c) and (d-ii) and the enlarged view of (d-ii) is 250 μm. (e) One section from a combined “virtual” two-tracer dataset generated by warping AAV-GFP brain onto CTB-Alexa-488 brain (see Supplementary Video 8). (f) Brain region marked up in (e) comprising motor cortex (M1) with overlapping anterograde (AAV-GFP, red color) and retrograde (CTB-Alexa-488, green color) labeling.