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SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction


We report a water-based optical clearing agent, SeeDB, which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. Our method maintained a constant sample volume during the clearing procedure, an important factor for keeping cellular morphology intact, and facilitated the quantitative reconstruction of neuronal circuits. Combined with two-photon microscopy and an optimized objective lens, we were able to image the mouse brain from the dorsal to the ventral side. We used SeeDB to describe the near-complete wiring diagram of sister mitral cells associated with a common glomerulus in the mouse olfactory bulb. We found the diversity of dendrite wiring patterns among sister mitral cells, and our results provide an anatomical basis for non-redundant odor coding by these neurons. Our simple and efficient method is useful for imaging intact morphological architecture at large scales in both the adult and developing brains.

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Figure 1: Optical clearing using SeeDB.
Figure 2: Morphological and chemical stability.
Figure 3: Fluorescence imaging of adult mouse brain.
Figure 4: Topographic organization of callosal axon pathways revealed by in utero electroporation and optical clearing with SeeDB (SeeDBp protocol).
Figure 5: Distribution of mitral cell somata labeled from a single glomerulus.
Figure 6: Tracing lateral dendrites of sister mitral cells.

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We thank J.R. Sanes (Harvard) (Thy1-YFP-G, H) and P. Mombaerts (Max Planck Institute) (OMP-GFP) for providing mouse strains. We are also grateful to M. Eiraku and K. Muguruma for assistance with two-photon microscopy setup, Olympus for the customized objective lens, Y. Mimori-Kiyosue for equipment, J. Nabekura and T. Nemoto for instructions on in vivo two-photon imaging, and R. Iwata and H. Hiraga for valuable comments on the manuscript. This work was supported by grants from the PRESTO program of the Japan Science and Technology Agency, the Sumitomo Foundation, the Nakajima Foundation, the Mitsubishi Foundation, the Strategic Programs for R&D (President's Discretionary Fund) of RIKEN, and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (to T.I.). The imaging experiments were supported by the RIKEN Center for Developmental Biology Imaging Facility and the Four-dimensional Tissue Analysis Unit. Animal experiments were supported by the Laboratory for Animal Resources and Genetic Engineering at the RIKEN Center for Developmental Biology.

Author information

Authors and Affiliations



M.-T.K. performed most of the experiments. S.F. performed in utero electroporation. T.I. conceived the experiments, performed the initial phase of experiments, supervised the project and wrote the manuscript.

Corresponding author

Correspondence to Takeshi Imai.

Ethics declarations

Competing interests

M.-T.K. and T.I. hold a patent (pending) for the SeeDB technique.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–5, Supplementary Figures 1–13, Supplementary Videos 1–14 (PDF 1863 kb)

Supplementary Video 1

Adult Thy1-YFP-H line (P70) imaged using confocal microscopy. Serial x-y images were taken at an interval of 10 μm. Depths (z) indicated in the movie are non-calibrated values. The real depths are 1.49× larger. See legend for Figure 3a. (AVI 1814 kb)

Supplementary Video 2

Volume rendering of adult Thy1-YFP-H mouse (P72) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from dorsal side, and 14×1 blocks were tiled. See legend for Figure 3c. (AVI 4780 kb)

Supplementary Video 3

Serial horizontal optical sections of Thy1-YFP-H line (P72). Hemi-brain sample was cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from dorsal side every 500 μm. See legend for Figure 3d. (AVI 2034 kb)

Supplementary Video 4

Thy1-YFP-H mouse (P72) imaged using two-photon microscopy. Serial x-y images were taken at an interval of 25 μm. See legend for Figure 3e. (AVI 2726 kb)

Supplementary Video 5

Volume rendering of serial optical sections shown in Supplementary Video 2. See legend for Figure 3e. (AVI 2058 kb)

Supplementary Video 6

Volume rendering of adult Thy1-YFP-H line (P72) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from medial side, and 14×1 blocks were tiled. See legend for Suplementary Figure 10a. (AVI 4410 kb)

Supplementary Video 7

Serial optical sections of Thy1-YFP-H line (P72) hemi-brain imaged from medial face using two-photon microscopy. Images were taken from the medial side every 300 μm, and 35×17 blocks were tiled. See legend for Supplementary Figure 10b. (AVI 2080 kb)

Supplementary Video 8

Thy1-YFP-H line (P21) cleared with SeeDB37 and imaged using two-photon microscopy. Images were taken from the dorsal side, and serial x-y images are shown. See legend for Supplementary Figure 11c. (AVI 6362 kb)

Supplementary Video 9

Volume rendering of serial optical sections shown in Supplementary Video 8. See legend for Supplementary Figure 11c. (AVI 5710 kb)

Supplementary Video 10

The entire olfactory bulb of a Thy1-YFP-G mouse (P21) imaged using two-photon microscopy. Serial tiled x-y images were taken from the medial side. See legend for Supplementary Figure 12. (AVI 2633 kb)

Supplementary Video 11

Callosal axons imaged using two-photon microscopy. Serial x-y images were taken at an interval of 10 μm. See legend for Figure 4. Left, anterior; Right, posterior. (AVI 12430 kb)

Supplementary Video 12

Topographic organization of corpus callosum imaged using two-photon microscopy. Anterior and posterior regions of the cerebral cortex layer II-III neurons were labeled with tdTomato (magenta) and EGFP (green), respectively, using in utero electroporation. Serial x-y images were taken at an interval of 15 μm. InSight DeepSee Dual (Spectra Physics) was used to excite tdTomato and EGFP at 1,040 nm and 920 nm, respectively. Our customized 25× objective lens was used. Left, anterior; Right, posterior. (AVI 13490 kb)

Supplementary Video 13

Reconstruction of individual callosal axons sparsely labeled by Cre-loxP system. See legend for Figure 4b. (AVI 2812 kb)

Supplementary Video 14

Tracing lateral dendrites of sister mitral cells. Serial confocal images to reconstruct Figure 6a. Depths (z) in the movie are non-calibrated values. The real depths are 1.49× larger. Only presumptive mitral cells were reconstructed in Figure 6a. See legend for Figure 6. Green, OMP-GFP; magenta, Alexa-647 dextran. (AVI 3144 kb)

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Ke, MT., Fujimoto, S. & Imai, T. SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16, 1154–1161 (2013).

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