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


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.

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Figure 1: STP tomography.
Figure 2: Retrograde tracing by CTB–Alexa Fluor 488.
Figure 3: Anterograde tracing by AAV-GFP and brain warping.

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We thank Y. Bao and R. Palaniswamy for expert technical assistance, S.C. Chen and M. Culpepper for design of the flexure-based microtome, Z.J. Huang (Cold Spring Harbor Laboratory) for the Sst-IRES-CreAi9 mouse, W. Denk, K. Rockland and F. Scalia for critical reading of the manuscript, and J. Kuhl for preparing art graphics and animation. This work was supported by Simons Foundation grant (137480) to P.O., McKnight Technological Innovations in Neuroscience Award to P.O., Howard Hughes Medical Institute Collaborative Innovation ward 43667 to H.S.S., and US National Institutes of Health grants 1 R43 HL093897-01 to T.R., R43 CA097670-01 and R44 CA097670-02 to T.R., K.B. and J.S.

Author information

Authors and Affiliations



J.S., T.R., K.B. and P.O. planned the redesign of the instrument; T.R., J.S. and K.B. engineered and built the instrument; T.R. and J.S. wrote the operating software; L.R.K. did all imaging experiments; J.T. contributed to early imaging experiments; Y.K. validated warping accuracy; K.U.V. and I.A.-C. set up all image-processing methods; H.S.S. supervised image processing; T.R. and K.B. contributed to writing instrument portions of the paper; and P.O. supervised imaging, coordinated all work and wrote the paper.

Corresponding author

Correspondence to Pavel Osten.

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Competing interests

K.B. and T.R. are shareholders and employees of TissueVision, Inc.; J.S. is an employee of TissueVision Inc.; and P.O. is a consultant of TissueVision Inc. and a shareholder and consultant of Certerra Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 (PDF 36829 kb)

Supplementary Video 1

STP tomography. The schema shows a representation of the instrument, including the x, y, and z stages, water bath with brain sample and vibratome. Optical excitation path from Ti:sapphire laser is drawn as a dashed red line; the laser beam is scanned using galvanometric x- and y-axis mirrors; fluorescence from the sample is collected via an optical detection path, drawn as a dashed green light and detected with photomultiplier tube (PMT). The video (after pressing the 'play' button) shows a zoom-in view of the water bath during imaging and cutting of a brain sample, with a simultaneous mosaicing of a coronal GFPM brain section. (SWF 7544 kb)

Supplementary Video 2

260 coronal section datasets showing 3D reconstruction of the GFPM mouse brain. The STP tomography 2D brain images were downsampled by factor of 5. (MOV 19165 kb)

Supplementary Video 3

260 coronal section datasets showing 3D reconstruction of the Mobp-GFP mouse brain. The STP tomography 2D brain images were downsampled by factor of 5. (MOV 18912 kb)

Supplementary Video 4

260 coronal section datasets showing 3D reconstruction of the Chat-GFP mouse brain. The STP tomography 2D brain images were downsampled by factor of 5. (MOV 16933 kb)

Supplementary Video 5

260 coronal section datasets showing 3D reconstruction of Sst-IRES-Cre::Ai9 mouse brain. The STP tomography 2D brain images were downsampled by factor of 5. (MOV 19604 kb)

Supplementary Video 6

800 coronal section dataset showing 3D reconstruction of olfactory bulbs from the Sst-IRES-Cre::Ai9 mouse brain, imaged at 2.5μm z-resolution. The STP tomography 2D images were downsampled by factor of 5. (MOV 11721 kb)

Supplementary Video 7

The same Sst-IRES-Cre::Ai9 olfactory bulb dataset as shown in Supplementary Video 6 after optical reslicing in y-z dimension at isotropic 2.5μm × 2.5μm × 2.5μm. (MOV 21707 kb)

Supplementary Video 8

AAV-GFP brain was warped onto CTB–Alexa-Fluor-488 brain and the datasets are shown as overlays in red and green color, respectively. The 2D brain images were downsampled by factor of 20 before warping. (MOV 3620 kb)

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Ragan, T., Kadiri, L., Venkataraju, K. et al. Serial two-photon tomography for automated ex vivo mouse brain imaging. Nat Methods 9, 255–258 (2012).

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