Array Tomography

Array tomography (AT) is a new high-throughput proteometric imaging method offering unprecedented capabilities for high-resolution imaging of tissue molecular architectures.  AT is based on (1) automated  ultrathin physical sectioning of resin-embedded tissue specimens, (2)  construction of planar arrays of serial ultrathin sections on optical coverslips, (3) staining and imaging of these  arrays, and (4) computational reconstruction into three dimensions, followed by (5) volumetric image analysis.  Array can be imaged by immunofluorescence or by scanning electron microscopy, or both.  Because these arrays are very effectively stabilized by the glass substrate, they can withstand many repeated cycles of staining, imaging and elution.  This permits the imaging of large numbers (i.e., 20 or more) of antibodies in addition to ultrastructure on each individual section.  [Movies]


Methodology:

Tapia, J.C., Kasthuri, N., Hayworth, K., Schalek, R., Lichtman, J.W., Smith, S.J and Buchanan, J.  (2012) High contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy.  Nature Protocols 7:193-206.

Micheva, K.D., Busse, B.L., Weiler, N.C., O'Rourke, N., Smith, S.J (2010) Single-synapse analysis of a diverse synapse population: Proteomic imaging methods and markers. Neuron 68:639-653 

Micheva, K.D., O’Rourke, N., Busse, B., and Smith, S.J (2010) Array Tomography: High-Resolution Three-Dimensional Immunofluorescence.  In: Imaging: A Laboratory Manual, 3rd Ed.  Cold Spring Harbor Press, Ch. 45, pp. 697-719. 

Micheva, K.D., and Smith, S.J (2007)  Array tomography: A new tool for imaging the molecular architecture and ultrastructure of neural circuits.  Neuron 55:25-36.

Applications:

Allen, N.J, Howe, M.L., Foo, L.C., Wang, G.X., Chakraborty, C., Smith, S.J and Barres, B.A. (2012)  Astrocyte-derived glypicans 4 and 6 promote the formation of excitatory synapses containing GluA1 AMPA glutamate receptors.  Nature (in press).

Saatchi S, Azuma J, Wanchoo N, Smith SJ, Yock PG, Taylor CA, Tsao PS. (2012) Three-dimensional microstructural changes in murine abdominal aortic aneurysms quantified using immunofluorescent array tomography. J Histochem Cytochem. 60:97-109.

Kleinfeld, D., Bharioke, A., Blinder, P., Bock, D.D., Briggman, K.L., Chklovskii, D.B., Denk, W., Helmstaedter, M., Kaufhold, J.P., Lee, W.C., Meyer, H.S., Micheva, K.D., Oberlaender, M., Prohaska, S., Reid, R.C., Smith, S.J, Takemura, S., Tsai, P.S. and Sakmann, B. (2011) Large-scale automated histology in the pursuit of connectomes. J Neurosci. 31:16125-38.

Robles, E., Smith, S.J and Baier H. (2011) Characterization of genetically targeted neuron types in the zebrafish optic tectum.  Frontiers Neural Circuits 5:1-14.

Appelbaum, L., Wang, G., Yokogawa, T., Skariah, G.M., Smith, S.J, Mourrain, P. and Mignot, E. (2010) Circadian and homeostatic regulation of structural synaptic plasticity in hypocretin neurons. Neuron 68:87-98.

Li, L., Tasic, B., Micheva, K.D., Ivanov, V.M., Spletter, M.L., Smith, S.J, Luo, L. (2010) Visualizing the distribution of synapses from individual neurons in the mouse brain.  PLoS One 5(7):e11503.

Appelbaum, L., Wang, G.X., Maro, G.S., Mori, R., Tovin, A., Marin, W., Yokogawa, T., Kawakami, K., Smith, S.J., Gothilf, Y., Mignot, E. and Mourrain, P. (2009) Sleep-wake regulation and hypocretin-melatonin interaction in zebrafish. Proc Natl Acad Sci U S A 106(51):21942-7.

Datwani, A., McConnell, M.J., Kanold, P.O., Micheva, K.D., Busse, B., Shamloo, M., Smith, S.J and Shatz, C.J. (2009)  Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity. Neuron 64:463-70.

Eroglu, C., Allen, N.J., Susman, MW, O'Rourke, N.A., Park, C.Y., Ozkan, E., Chakraborty, C., Mulinyawe, S.B., Annis, D.S., Huberman, A.D., Green, E.M., Lawler, J., Dolmetsch, R., Garcia, K.C., Smith, S.J, Luo, Z.D., Rosenthal, A., Mosher, D.F. and Barres, B.A. (2009) Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis. Cell 139:380-92.

Koffie, R.M., Meyer-Luehmann, M., Hashimoto, T., Adams, K.W., Mielke, M.L., Garcia-Alloza, M., Micheva, K.D., Smith, S.J, Kim, M.L., Lee, V.M., Hyman, B.T., and Spires-Jones, T.L. (2009) Oligomeric amyloid beta associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques.  Proc. Natl. Acad. Sci., USA 106: 4012-7.

Stevens, B., Allen, N.J., Vazquez, L.E,. Howell, G.R.,Christopherson, K.S., Nouri, N., Micheva, K.D., Mehalow, A., Huberman, A.D., Stafford, B., Sher, A., Litke, A.M., Lambris, J.D., Smith, S.J., John, S.W.M., & Barres, B.A. (2007) The classical complement cascade mediates CNS synapse elimination. Cell 131:1164-78.

Commentaries:

O’Rourke, N.A., Weiler, N.C., Micheva, K.D. and Smith, S.J (2012) Deep molecular diversity of mammalian synapses: Why it matters and how to measure it.  Nature Reviews Neuroscience 13:365-379.

Micheva, K.D. and Bruchez, M.P. (2012) The gain in brain: novel imaging techniques and multiplexed proteomic imaging of brain tissue ultrastructure. Curr Opin Neurobiol 22:94-100.

Lichtman, J.W. and Smith, S.J (2008)  Seeing Circuits Assemble.  Neuron 60:441-448.

Smith, S.J (2007) Circuit Reconstruction Tools Today.  Curr. Opin. In Neurobiol. 17:601-608.


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Array tomography of mouse  somatosensory cortex, tangential section.  Download large bitmaps via links below:

Three-Color 3904x12528 pixels
mMos3_ThreeColor.tiff
(142 MB)

YFP only 3904x1258 pixels
mMos3_Grayscale.tif
(49 MB)

Layer 5b YFP 1920x1080 pixels
MAX_mMos3_YFP_L5b.tif
(2.1 MB)

Layer 5-6 YFP 3904x4208 pixels
MAX_mMos3_YFPL_5-6.tif
(16.4 MB)