A platform for brain-wide imaging and reconstruction of individual neurons
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- National Institute of Mental Health, United States
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
Figures
Figure 1 with 1 supplement
Schematic of imaging system.
(a) Schematic of apparatus for automated volumetric two-photon tomography. (b) To image large volumes of tissue, a collection of three-dimensional image stacks (tiles) covering the full volume of …
Figure 1—figure supplement 1
Point spread function measurement.
(a) Empirically-measured point spread function measured using 200-nm fluorescent polystyrene beads.
Figure 2
Sample preparation and clearing.
(a) Whole brains (left) and 1 mm-thick tissue sections (right) cleared using dimethyl sulfoxide (DMSO) and d-sorbitol. (b) Fluorescence of purified eGFP as a function of DMSO concentration (v/v) in …
Figure 3 with 1 supplement
Registration of image tiles.
(a) Example registration of pairs of image tiles in the axial (left) and lateral (right) directions. (b) Initial displacement of automatically-identified features as a result of sectioning (left) …
Figure 3—figure supplement 1
Lipofuscin imaging.
Overlay of fluorescence captured in the green (500–550 nm) and orange (580–653 nm) spectral bands from a representative tile within the neocortex. Autofluorescent lipofuscin (white puncta) could be …
Figure 4 with 1 supplement
Whole-brain imaging.
(a) Three-dimensional rendering of complete mouse brain dataset as viewed from an anterolateral (left) and ventral (right) perspective. (b) Maximum intensity projection through a large tissue volume …
Figure 4—figure supplement 1
High speed, high-power imaging.
(a) Image of an axonal collateral with low (top) and high (bottom) excitation power. High-power imaging improves signal-to-noise without degrading resolution due to fluorophore saturation. (b) …
Figure 5 with 1 supplement
Axon collaterals are labeled with high signal-to-noise across their entire length.
(a) Top: Virtual coronal section through whole-brain dataset. Boxed area denotes region containing labeled somata and is expanded in schematic below. Bottom: Laminar distribution and dendritic …
Figure 5—figure supplement 1
Signal-to-noise of axonal imaging.
(a) Representative images before (left) and after (middle) lossy H.264 compression. The intensity profile across each neurite (along paths denoted by dotted lines in images on left) is plotted to …
Figure 6
Complete reconstruction of axonal morphology.
(a) Complete reconstruction of the same five projection neurons depicted in Figure 4 (inset). Reconstructions are overlaid on a horizontal (left) and sagittal (right) outline of the imaged mouse …
Figure 7
Fine scale topology of contralateral cortical projections.
(a) Horizontal view of the dendrites and axons of two layer V IT neurons (red and black). Axons projecting to locations other than the contralateral motor cortex are shown in lighter colors. (b) …
Videos
Video 1
Movie illustrating 18 serially acquired image tiles that have been registered and resampled into a continuous image volume.
This volume (3 × 1 × 6 tiles) spans five adjacent tissue sections. Fine axons are resolvable and all fibers appear continuous. Images were spectrally unmixed to remove autofluorescence of lipofuscin.
Video 2
Single axon traced to its terminus.
Depicted path represents the longest continuous axonal segment starting at the cell soma. The terminus is located in the anterior piriform cortex. White dot corresponds to the same location in both …
Video 3
Three-dimensional rendering of reconstructed Layer II motor cortical neurons.
Displayed brain outline corresponds to the contours of the imaged brain. Color code is the same as in all figures.
Video 4
Three-dimensional rendering of reconstructed Layer V motor cortical neurons.
Displayed brain outline corresponds to the contours of the imaged brain. Color code is the same as in all figures.
Video 5
Three-dimensional rendering of reconstructed Layer VI motor cortical neuron.
Displayed brain outline corresponds to the contours of the imaged brain. Color code is the same as in all figures.
Tables
Table 1
Clearing solutions.
| Solution (#) | Dimethyl sulfoxide (g) | PB (g) | D-sorbitol (g) | Refractive index |
|---|---|---|---|---|
| 1 | 26.83 | 73.17 | 0.00 | 1.373 |
| 2 | 52.38 | 47.62 | 0.00 | 1.412 |
| 3 | 44.52 | 40.48 | 15.00 | 1.425 |
| 4 | 36.67 | 33.33 | 30.00 | 1.440 |
| 5 | 34.25 | 20.75 | 45.00 | 1.468 |
| 6 | 25.56 | 11.44 | 63.00 | 1.489 |
Table 2
Axonal reconstructions. All lengths in mm.
| Soma location | Layer II | Layer II | Layer V | Layer V | Layer VI |
|---|---|---|---|---|---|
| Dendritic branches | 66 | 57 | 37 | 25 | 23 |
| Dendritic length | 8.03 | 7.67 | 9.17 | 5.06 | 4.20 |
| Axonal branches | 79 | 67 | 178 | 136 | 27 |
| Axonal length | 47.34 | 41.04 | 121.20 | 75.44 | 23.57 |
| Axonal targets (ipsilateral) | Motor cortex Dorsal striatum Nucleus accumbens | Motor cortex Orbital cortex Dorsal/medial striatum Caudal/lateral striatum | Motor cortex Somatosensory cortex Auditory cortex Orbital cortex Agranular insular cortex Ectorhinal cortex Piriform cortex Dorsal striatum Nucleus accumbens Olfactory tubercle Taenia tecta | Motor cortex Somatosensory cortex Auditory cortex Anterior cingulate cortex Posterior parietal cortex | Motor cortex Thalamic nuclei: Ventral anterior lateral Posterior Submedial Reticular |
| Axonal targets (contralateral) | Motor cortex Insular cortex Piriform cortex | Motor cortex Anterior cingulate cortex Agranular insular cortex Claustrum Basolateral amygdala | Motor cortex Piriform cortex Dorsal striatum Nucleus accumbens Olfactory tubercle Taenia tecta | Motor cortex Anterior cingulate cortex Dorsal striatum |
Table 3
Comparison with alternative technologies for whole-brain imaging.
| Method | Lateral resolution (L; µm) | Axial resolution (A; µm) | L × L × A (µm3) | Speed (× 106 µm3/s) |
|---|---|---|---|---|
| This study | 0.45 | 1.33 | 0.26 | 1.6 |
| Selective plane illumination microscopy* | 0.65 | 7.30 | 3.10 | 160 |
| Knife-edge scanning microscopy/ micro-optical sectioning tomography (Li et al., 2010) | 0.71 | 1.0 | 0.50 | 1.0 |
| Transmission electron microscopy with camera array (Bock et al., 2011) | 0.004 | 0.045 | 7.2 x 10-7 | 5.6 x 10-6 |
| Serial block-face electron microscopy (Helmstaedter et al., 2013) | 0.0165 | >0.025 | >6.8 x 10-6 | 1.1 x 10-6 |
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*Ideal resolution in fully cleared whole mouse brain (P. Keller, personal communication).
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Comparison of resolution and imaging speed with other imaging modalities. Resolution values represent full width at half maximum except for in transmission electron microscopy with camera array and serial block-face electron microscopy, where pixel sizes are reported.
