Skip to main content
. 2022 Oct 11;11:e77419. doi: 10.7554/eLife.77419

Figure 2. High refractive index imaging buffer EZ View achieves deeper imaging depth and maintains fluorescence stability.

Figure 2.

(A) Refractive index (RI) of 80% Nycodenz increases linearly with increasing concentrations of urea (n=3). (B–D) Comparison of transparency of adult mouse brains processed with EZ Clear lipid removal, washing, and then equilibration in (B) PBS (RI = 1.332), (C) refractive index matching solution (RIMS) (RI = 1.463), and (D) EZ View (RI = 1.518) for 24 hr at room temperature. Comparison of 3.5-month-old mouse brains perfused with lectin-649, then treated with delipidation, water washes, equilibrated in (E–G) RIMS and (H–J) EZ View, and imaged by lightsheet fluorescence microscopy (LSFM). (E and H) Comparison of volume rendered whole mouse brains, transverse view, and (F and I) across the imaging axis starting from dorsal to ventral, and (G and J) color-coded depth projection at 1 mm intervals beginning from dorsal (0 mm) to ventral (6 mm) side. (K) Quantitative comparison of mean fluorescence intensity of lectin-694 at different imaging depths (dorsal to ventral) shows the signal intensity gradually decreases along with the imaging depth when the brains were equilibrated in RIMS, unlike those equilibrated in EZ View. (n=3, error bars represent standard deviation [SD]. Two-way ANOVA and multiple comparisons. ns – not significant, **p<0.01, ***p<0.001.) (I) The fluorescence intensity of lectin-694 remain stable when stored in EZ View up to 70 days (n=3, two-way ANOVA and multiple comparisons).

Figure 2—source data 1. Measurements for refractive index changes in response to different concentrations of Nycodenz and urea, quantitative comparison of fluorescence intensity of lectin-649 at different imaging depths between brains equalibrated in EZ View and RIMS, and fluorescence intensity measurement of lectin-649 after storing samples in EZ View for 14, 17, 43, and 70 days.