Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains

PLoS One. 2015 May 20;10(5):e0124650. doi: 10.1371/journal.pone.0124650. eCollection 2015.

Abstract

In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Propanol / chemistry
  • Animals
  • Brain / cytology*
  • Brain / virology
  • Brain Mapping / methods*
  • Cell Line
  • Cricetinae
  • Green Fluorescent Proteins
  • Imaging, Three-Dimensional / methods*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Microscopy, Fluorescence / methods*
  • Neurons / cytology*
  • Neurons / virology
  • Rabies virus
  • tert-Butyl Alcohol / chemistry

Substances

  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • 1-Propanol
  • tert-Butyl Alcohol

Grant support

This work was supported by Grants SCH 15578/1-1 and SFB 1089 (P02) by Deutsche Forschungsgemeinschaft (www.dfg.de) to MKS and by general funding by the Max-Plank Society, Germany (www.mpg.de) to GG, AS, MKS, and RS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.