Optimized single-step optical clearing solution for 3D volume imaging of biological structures

Abstract

Various optical clearing approaches have been introduced to meet the growing demand for 3D volume imaging of biological structures. Each has its own strengths but still suffers from low transparency, long incubation time, processing complexity, tissue deformation, or fluorescence quenching, and a single solution that best satisfies all aspects has yet been developed. Here, we develop OptiMuS, an optimized single-step solution that overcomes the shortcomings of the existing aqueous-based clearing methods and that provides the best performance in terms of transparency, clearing rate, and size retention. OptiMuS achieves rapid and high transparency of brain tissues and other intact organs while preserving the size and fluorescent signal of the tissues. Moreover, OptiMuS is compatible with the use of lipophilic dyes, revealing DiI-labeled vascular structures of the whole brain, kidney, spleen, and intestine, and is also applied to 3D quantitative and comparative analysis of DiI-labeled vascular structures of glomeruli turfs in normal and diseased kidneys. Together, OptiMuS provides a single-step solution for simple, fast, and versatile optical clearing method to obtain high tissue transparency with minimum structural changes and is widely applicable for 3D imaging of various whole biological structures.

Fig. 1. OptiMuS outperforms other optical clearing methods when considering transparency, clearing rate, and size preservation together.

a The timelines of OptiMuS, CLARITY, CUBIC, ScaleS, ScaleSQ(0), SeeDB2G, FOCM, and MACS for clearing of 1-mm thick rat brain sample. b Bright-field images of pre-and post-cleared by each method in a. Overlapped images of outlined pre- and post-cleared brain tissues (black: pre-, red: post-cleared). Grid size = 1.5 × 1.5 mm. c Quantitative comparison of the linear expansion after processing with each clearing method (n = 6). d Transmittance scan curves at 400–800 nm after clearing 1-mm thick rat brain samples by each method (n = 4). e Transmittance at 600 nm vs. size change graph for each method. The data were shown as the mean ± SD. ***p < 0.001, **p < 0.01, *p < 0.05.

Fig. 2. OptiMuS preserves the fluorescence signal of endogenous proteins.

a Fluorescence images of the endogenous EYFP signals of 50-μm thick Thy1-EYFP brain slices were taken daily at the same position after clearing with each method. Scale bar = 100 µm. b Normalized fluorescence intensity curves of images taken daily after each clearing method, normalized to the signals on day 0. The data were shown as the mean ± SD (n = 3). ***p < 0.001, **p < 0.01, *p < 0.05. c Fluorescence images of 1-mm thick Thy1-EYFP brain slices at various depths after clearing with OptiMuS and PBS for 1.5 h. Scale bar = 200 µm. d SNR values over the depth of imaging in c (n = 3).

Fig. 3. OptiMuS enables 3D visualization of neural structure networks.

a Top view of maximum intensity z-projection image of the whole brain from Thy1-EYFP transgenic mouse cleared by OptiMuS for 3 days. Scale bar = 2 mm. b–d Maximum intensity z-projection images of the optical sections between 0 and 2 mm (b), 2–4 mm (c), and 4–6 mm (d). Scale bar = 2 mm. Parts of magnified 3D projection images and optical section images at 500, 1000, and 1500 µm of the cortex (e) and striatum (f) in a, respectively. Scale bar = 300 µm, 500 µm respectively. g 3D reconstruction image of the intestine from ChAT-Cre-tdTomato transgenic mouse after OptiMuS clearing for 10 hr. Scale bar = 1 mm. h Magnified maximum z-projection image of the area enclosed by a rectangle in g. The networks of cholinergic neurons including the myenteric plexus and submucosal plexus were clearly visualized. Scale bar = 200 µm. i 3D reconstruction images of the nucleus accumbens (NAc) region of ChAT-Cre-tdTomato mouse brain after OptiMuS clearing for 1 h. The 3D axis is expressed in µm. j Optical section images at each depth. Scale bar = 100 µm.

Fig. 4. OptiMuS visualizes DiI-labeled 3D vascular structures of various whole organs.

a The schematic diagram of DiI-labeled whole and axially sectioned brain imaging with LSFM after OptiMuS clearing for 3.5 days. b 3D reconstruction image of DiI-labeled whole mouse brain. c Top view of maximum intensity z-projection image of DiI-labeled mouse brain that was halved axially (See a). Scale bar = 2 mm. d–f Magnified images of the areas enclosed by rectangles in c. Scale bar = 500 µm. g–h The schematic diagram and 3D reconstruction image of DiI-labeled coronally sectioned brain imaging with LSFM after OptiMuS clearing for 3.5 days. i 3D reconstruction images of the vascular structures of DiI-labeled whole intestine (i) and spleen (k). Scale bar = 1 mm (g) and 2 mm (h). j, l Pseudo-color depth-coded images of the boxed regions in g and h, respectively.

Fig. 5. OptiMuS enables comparative 3D analysis of glomeruli structures of normal and diseased kidneys.

a The workflow for the comparative 3D analysis of DiI-labeled kidney glomeruli structures via OptiMuS and DXplorer. b 3D reconstruction image of DiI- labeled whole mouse kidney after OptiMuS clearing and LSFM imaging. Scale bar = 2 mm. c Magnified view of the boxed region in b. Scale bar = 250 µm. d 3D reconstruction image of a single kidney glomerulus in c. Scale bar = 50 µm. e Representative 3D mesh images of individual glomeruli of normal and NTN model. f 3D features including volume, hMAX, and nMAX were extracted from the 3D meshes of the normal glomeruli and NTN glomeruli and analyzed by DXplorer. hMax (maximum head diameter), and nMax (maximum neck diameter). n = 18 (normal) and 10 (NTN). The data are shown as the mean ± SD ***p < 0.001.