Resolution characterization and image depth of standard adapter and RIM-Deep.

(A and B) Schematic diagram of a 10X immersion objective with a standard adapter (A) or RIM-Deep (B).

(C-D) MIP of 3-μm-diameter beads imaged in the xy and yz planes using the standard adapter (C) or RIM-Deep (D) at different axial positions.

(E) Axial resolutions for a 10X immersion objective paired with standard adapter or RIM-Deep at different axial positions. The resolution is estimated by FWHMs of intensity profiles with a Gaussian fit for 3-μm-diameter beads embedded in 1% agarose dissolved in CLARITY mounting solution (with more than 10 beads per plane). Data are presented as mean ± s.e.m.

(F) The experimental scheme for the brain clearing process in Macaca fascicularis.

(G, H, I) Three-dimensional reconstruction of the Macaca fascicularis brain vasculature using three different tissue clearing methods (iDISCO, CUBIC or MACS) with a standard adapter. (G0, H0, I0) MIP of G, H or I in xz plane. (G1-G2, H1-H2, I1-I2) Optical section of (G, H, I) at varying depths.

(J, K, L) Three-dimensional reconstruction of the Macaca fascicularis brain vasculature using iDISCO, CUBIC or MACS) with a RIM-Deep. (J0, K0, L0) MIP of J, K or L in xz plane. (J1-J4, K1-K4, L1-L4) Optical section of (J, K, L) at varying depths.

Evaluation of imaging depth for a Thy1-EGFP mouse brain using RIM-Deep.

(A) Experimental scheme.

(B) 3D reconstruction of the ∼5 mm deep in the mouse brain (left), MIP of xz side view (middle) and MIP of yz side view (right).

(C and G) 3D reconstructions of neuronal structures within the hippocampus (yellow box) and thalamus (orange box), respectively, as indicated in B.

(D and H) Lateral slices through the indicated lateral planes in (C and G). Zoom-in views of the selected areas in top right.

(E-F) MIP of xy side view and xz side view of C. (I-J) MIP of xy side view and yz side view of G.

3D imaging and reconstruction of neural and vascular structures in intact tissues using RIM-Deep.

A) 3x3 stitching pattern of deep imaging of a cleared brain.

(B) MIP of yz side view of A.

(C) Optical section of top layer in (B). Zoom-in views of the selected areas in top right.

(D) 3D reconstruction of a half of cleared brain in a Thy1-eGFP mouse brain. The white box represents MIP.

(E-I) Stitched single layer images in the Z-direction.

(J) 3D imaging of the entire brain vasculature in ischemic stroke mice. The images along the z stack are colored by spectrum.

(K) MIP of vascular imaging in the ischemic region (red box in J).

(L) MIP of vascular imaging in the contralateral region (blue box in J).

Illustration and actual picture of standard adapter (A) and the RIM-Deep (B) mounted on the Nikon AXR microscope.

Design and configuration of RIM-Deep for inverted confocal microscope.

(A) A Three-view diagram of a media reservoir.

(B) 1-3, Three-view diagram of the support bracket. 4, Three-dimensional diagram of the specimen holder.

(C-F) RIM-Deep assembly procedure.

High-depth imaging using Leica STELLARIS 5 with RIM-Deep.

(A) Setup of Leica STELLARIS 5 microscope with RIM-Deep assembly.

(B) 3D imaging of cleared Macaca fascicularis brain tissue.

(C) Optical sections of (B) at varying depths.