Figure 1

HyPer-3D abolishes tissue AF and increases optical transparency

(A) AF of the kidney, heart, and spleen imaged in the same field of view.

(B) Green AF exhibited by a single kidney cut into two halves, with one-half treated with HyPer-3D (HyP) and the other serving as an untreated control (Cont).

(C) Red AF exhibited by the untreated control (Cont) and HyPer-3D (HyP)-treated kidneys.

(D) Time-dependent quenching of green and red AF in 1 mm thick murine heart sections.

(E) Quantification of the decrease in green and red AF in the kidney and the heart tissue with HyPer-3D treatment (∗∗∗∗p < 0.0001).

(F–I) Adjacent 0.5 mm thick sections from a single kidney pretreated with HyPer-3D (HyP) or left as untreated controls (Cont), and then processed with 3D clearing protocols, namely (F) EZ View (also see Video S3), (G) CUBIC-2 (also see Video S4), (H) MACS-R2 (also see Video S5), and (I) FOCM (also see Video S6). Images of the center region of HyP and Cont kidney sections are shown at different time intervals for comparison (see also Figure S2).

(J) Quantification of optical transparency of HyP and Cont tissues over time in refractive index-matching solutions, as determined by the underlying grid visibility.

(K–O) Adjacent 0.5 mm thick sections treated with HyPer-3D (HyP) or left untreated (Cont), and then incubated with (K) ScaleS, (L) ClearT², (M) sRIMS, (N) SeeDB, and (O) glycerol.

(P) Optical clearing of whole adult murine kidneys with CUBIC L/R + (N). Kidneys were cut into left and right halves, treated with HyPer-3D (HyP) or left untreated (Cont), and then processed with CUBIC L/R + (N).

(Q) Optical clearing of adult murine kidney and heart with the EZ Clear method. Kidneys were cut into left and right halves, treated with HyPer-3D (HyP) or left untreated (Cont), and then processed with EZ Clear.

(R) Optical clearing of whole adult brains using EZ Clear. Brains were divided into left and right hemispheres, treated with HyPer-3D (HyP) or left untreated (Cont), and then processed using EZ Clear.

(S) Quantification of optical transparency, as assessed by the underlying grid visibility, of tissues treated with standard published protocols (Cont) vs. counterpart tissues first pretreated with HyPer-3D (HyP) and then subsequently processed with standard published protocols (∗∗∗∗p < 0.0001; ∗∗∗p < 0.001; ∗∗p < 0.01). See also Table S1 in supplemental information for corresponding quantitative values and statics. All scale bars are 1 mm.

AF, autofluorescence.