Figures and data
RIM-Deep on an inverted confocal microscope for increased imaging depth of cleared tissues
(A and C) The 10X long working distance objective demonstrates significant losses in axial resolution due to the deleterious effects of mismatched refractive index.
(B and D) 10X immersion objective paired with RI adapter provides superior axial resolution throughout due to refractive index matching.
(E) Axial resolutions for a 10X long working distance objective and a 10X immersion objective paired with 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) Experimental scheme illustrates the process of brain structure visualization in Macaca fascicularis, involving fixation, immunolabeling, and optical clearing with iDISCO (RI=1.56).
(G) The official adapter displays a three-dimensional reconstruction of a cleared brain up to a depth of 2 mm.
(H) RIM-Deep showcases a deeper 3D reconstruction of the cleared brain, extending to za depth of 5 mm.
(I) Progressive 2D slices of the reconstruction in (G) at varying depths, ranging from 0.5 mm to 5 mm.
(J) A top-down view of the vascular network (left) and the maximum intensity projection (MIP) of the XZ side view (right) from (H).
RIM-Deep improves imaging depth at micron-level resolution in Thy1-EGFP transgenic mouse brain
(A) A Thy1-EGFP mouse brain undergoes CUBIC clearing for subsequent 3D imaging;
(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 and thalamus, 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.
The 3D imaging and reconstruction of neural and vessel structures in intact tissues
(A) 3×3 stitching pattern of deep imaging of a cleared brain;
(B) MIP of YZ side view of A;
(C) 2D planar image of (B). The magnified view in the top left white box distinctly illustrates the discernibility of neuronal somas and fibers.
(D) 3D reconstruction of a half cleared brain in a Thy1-eGFP mouse brain. The white box represents MIP;
(E-I) Selected 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;
(L) MIP of vascular imaging in the contralateral region.
Comparative illustration highlighting the distinctions between the RIM-Deep and the official adapter
(A) A conceptual illustration of cleared tissue imaging with standard adapter. Challenges remain with gravitational RI buffer dispersion between the objective and specimen, leading to RI mismatch.
(B) A conceptual illustration of cleared tissue imaging with the RI adapter, which ensures the stability and consistency of the refractive index during deep imaging.
Design and configuration of RIM-Deep for inverted confocal microscope
(A) A schematic diagram of a solution reservoir. ① A side view of the solution reservoir. ② A top view of the solution reservoir;
(B) A schematic diagram of a support bracket and specimen holder. ① A side view of the support bracket. ② Top view of the support bracket.③ The front view of the support bracket. ④Three-dimensional diagram of the specimen holder;
(C) The solution reservoir integrates with the cap;
(D) The specimen holder can be individually customized based on the size and morphological characteristics of the samples and is strategically positioned at the precise center of support bracket;
(E) The solution reservoir with the cap, affixed to the immersion objective, is designed to accommodate various imaging buffers;
(F) The specimen holder is placed on the stage through the adapter.
