Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.
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| 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.
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| Authors | Martin K Schwarz, Annemarie Scherbarth, Rolf Sprengel, Johann Engelhardt, Patrick Theer, Guenter Giese |
| Journal | PloS one (PLoS One) Vol. 10 Issue 5 Pg. e0124650 ( 2015) ISSN: 1932-6203 [Electronic] United States |
| PMID | 25993380 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
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