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Figure 1. Effectiveness of iHE. (A) Device configuration of iHE. (B) Effect of ultrasound on enhanced rinsing: the x-axis represents the rinsing time, and the y-axis represents the absorbance of the rinsing solution. The p value between the ultrasound and control groups showed a sharp decrease over time (Student’s t test, n = 5). After rinsing for 20 min, the difference between the ultrasound and control groups was significant (p < 0.001). Comparison of the two groups was performed by unpaired t-test (two tailed). All data are presented as the mean ± standard error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Enhancing the staining effect of DCM delipidation and ultrasound. C57BL/6 mouse brains were stained with hematoxylin and then were cut along the maximum sagittal plane. I With DCM delipidation but without ultrasound; staining for 6.5 h. II With DCM delipidation and with ultrasound; staining for 6 h. III Without DCM delipidation and without ultrasound; staining for 6.5 h. IV Without DCM delipidation but with ultrasound; staining for 6 h. All staining was performed at 50 °C.

Figure 2. Comparison of iHE and traditional H&E staining. (A–C), Images of 7-μm-thick mouse brain slices stained using the traditional H&E method. (D–F), Images of intact mouse brain tissues after iHE staining, slicing and 2D imaging. The red matter appearing in (A~F) represents blood cells that were not completely cleared during cardiac perfusion. Objective lens, 20×; N.A., 0.75; work distance, 1 mm. Scale bar: 50 μm.

Figure 3. Slices from an intact mouse brain stained with iHE, followed by 2D imaging. (A) 3D projection of 20 slices in (B). (B) The coronal plane slices from the C57BL/6 mouse brain after iHE. Objective lens, 4× and 20×; N.A., 0.2 and 0.75. The brain was sliced at the coronal plane for 8 μm, and we selected one slice for every 400 μm from the olfactory bulb to the epencephalon. (D–F) Magnification of (C).

Figure 4. Images of other mouse tissues stained with iHE. (A–C) Images of mouse liver with a tumor. (D,E) iHE for mouse lung. (F,G) iHE for mouse kidney. Objective lens, 20×; N.A., 0.75; work distance, 1 mm.

Figure 5. Mouse brain perfused with carbon ink before iHE. (A,B) Hippocampus, (B) is the magnification of the box in (A). (C,D) Thalami, (D) is the magnification of the box in (C). Objective lens, 20×; N.A., 0.75; work distance, 1 mm.

Figure 6. Possible principle of iHE. A fixed tissue is used as the original tissue. After dehydration, the tissue is subjected to delipidation, followed by the H&E staining procedure; the H&E staining process was simplified so that the reader could better understand the principle for how ultrasound is involved in the process. Dichloromethane can cause a porous state of the cell membrane, equal to the porous state of the tissue. The pores of the tissue change in the ultrasonic field because ultrasound is a longitudinal wave. Thus, the pores in the wave crest of the ultrasound are stretched, while the pores in the trough of the waves are compressed. Meanwhile, the random motion of particles is enhanced in the ultrasonic field. The diffusion process is enhanced and achieves rapid and uniform staining.

 Movie S1. GFP-labeled cholinergic neurons distributed throughout the entire cerebral cortex

Movie S2. The arborization of 50 well-separated cholinergic neurons in the MS/VDB throughout the brain

Movie S3. Individual cholinergic neurons can project long distances to distinct brain regions through different projection routes