(a) Whole brains (left) and 1 mm-thick tissue sections (right) cleared using dimethyl sulfoxide (DMSO) and d-sorbitol. (b) Fluorescence of purified eGFP as a function of DMSO concentration (v/v) in 10 mM HEPES, pH 7.3 (open circles) and 10 mM HEPES, pH 7.3 containing 44% (w/v) d-sorbitol (filled circles). (c) eGFP and tdTomato fluorescence in a cleared hemi-brain demonstrates preservation of native fluorescence. (d) Maximum intensity projections of side views of image tiles of histone 2B-eGFP labeled nuclei from a DMSO/d-sorbitol cleared section (right) and from a matched section in phosphate buffered saline (PBS; left) from the contralateral hemisphere of the same brain. (e) Quantification of intensities of histone 2B-eGFP labeled nuclei as a function of depth (PBS: n=204 detected nuclei in two tiles; DMSO/d-sorbitol: n=815 detected nuclei in three tiles). (f) Representative images of a single axonal collateral as it passes through the anterior commissure (white) and surrounding olfactory cortex (gray). Images were acquired at a depth >180 μm in each case and scaled in the same manner. (g) Axons labeled with multiple fluorophores could be simultaneously imaged with high signal to noise within cleared tissue. Note that imaging fine axons through the axially oriented anterior commissure in this example represent a stringent test of clearing due to the high degree of optical scattering observed in white matter tracts. DMSO, dimethyl sulfoxide; eGFP, enhanced green fluorescent protein.