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Staining, Tracing, and Imaging Techniques

開催日 2014/9/12
時間 11:00 - 12:00
会場 Poster / Exhibition(Event Hall B)

Three-dimensional reconstruction of ultrathin-sectioned brain by light microscopy

  • P2-387
  • 岩崎 広英 / Hirohide Iwasaki:1 岡部 繁男 / Shigeo Okabe:1 
  • 1:東京大院・医・神経細胞生物 / Dept Cellular Neurobiol., Graduate School of Medicine, Univ of Tokyo, Tokyo, Japan 

The brain consists of a huge number of neurons and they are connected each other via synapses to generate neuronal circuits. In order to understand how neuronal circuits generate the brain functions, it is important to obtain the full wiring diagram of neurons in the whole brain (connectome). However, the wiring diagram of the brain is highly complex and there are still severe technological limitations. One of these technological problems is that three-dimensional reconstruction of neuronal circuits requires high-resolution images to identify synapses but it should also cover relatively large imaging areas for drawing a complete map. In order to overcome this discrepancy, it is essential to combine the technology of both light microscopy and electron microscopy.
In this poster, we show our hybrid approach of electron and light microscopy by introducing ultrathin sectioning techniques for light microscopy. The limit of resolution in conventional light microscopy is 750~1μm along z-axis but ultrathin sectioning can overcome this limitation. We introduced both DsRed and PSD95-GFP into cortical neurons by using in utero electropolation. We next embedded the brain tissue that corresponds to a part of the somatosensory cortex in LRWhite resin. Because LRWhite is a hydrophilic resin, DsRed remains fluorescent after embedding. The ultrathin brain sections in LRWhite are suitable for immunostaining and we could visualize synapses by either anti-VGluT1 or anti-GFP antibodies. Serial sections with 250 nm of thickness were imaged by confocal microscopy to reconstruct three-dimensional structures. Sections were also observed by epifluorescence microscopy with an automated stage and images were obtained from multiple fields of view and stitched to cover a large area of the brain. These strategies are fundamental for revealing the wiring diagram of the brain.

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