Photoclearing of whole human organs.

(A) The mCleLight pipeline involves photogrammetry and clinical imaging to identify anatomical landmarks. The clearing protocol includes two main steps: chemical clearing to delipidate, decolorize, and permeabilize the tissue, followed by photoclearing with a high-power, wide-spectrum LED source to bleach pigments and autofluorescence. Cleared samples are then labeled, imaged using a light-sheet microscope, and processed for quantitative analysis. This panel was created using BioRender.com. (B) 3D volume reconstruction of a whole human heart with photogrammetry. Scale bars, 2 cm. (C) Surface volume rendering of a T1 MRI acquisition of the heart (right panel) and single transversal plane image extracted from the MRI volume (right panel). Scale bars, 2 cm. (D) Chemical clearing of the entire heart. A fabric was inserted inside large vessels and cavities to maintain its shape during clearing process. Grid scale 1 cm. (E) Heart after mCleLight photoclearing. Grid scale, 1 cm. (F) Single plane image from a 3D stack of the inset in panel E showing the pulmonary valve. The entire heart was labeled with Fast Green, while a weak residual autofluorescence signal was imaged to provide additional anatomical contrast. Scale bar 3000 µm. (G) Single plane image from a 3D stack of the inset in panel E showing the coronary arteries. Scale bar 500 µm. (H) Single plane image from a 3D stack of the inset in panel E. The ventricular myocardium was resolved. Scale bar 500 µm. (I) 3D volume reconstruction of a whole human kidney with photogrammetry. Scale bars, 2 cm. (J) Surface volume rendering of a T2 MRI acquisition of the kidney (left panel) and single transversal plane image extracted from the MRI volume (right panel). Scale bars, 2 cm. (K) Chemical clearing of the entire kidney. Grid scale 1 cm. (L) Kidney after mCleLight photoclearing. Grid scale, 1 cm. (M) The entire kidney was labeled with Fast Green and was imaged after mCleLight. Background autofluorescence signal was imaged to provide additional anatomical contrast. Scale bar, 2000 µm. (N) Higher magnification of the inset in panel K. Degenerated glomeruli were visualized by their spherical shape and fibrous composition labeled with Fast Green. Scale bar, 300 µm. (O) Single plane images form 3D stacks of a non-degenerated glomerulus (upper panel) and a degenerated glomerulus (lower panel). Scale bars 20 µm. (P) 3D volume segmentation in the renal cortex showing 20% of degenerated and 80% non-degenerated glomeruli. Scale bar, 500 µm.

Autofluorescence quenching across human organs.

(A) Liver 1cm-thick section before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (B) Imaging of autofluorescence levels after chemical clearing (upper rows) and after photoclearing (lower rows) across different excitation wavelengths (488, 555 and 647nm). The same imaging and image rendering parameters were used after chemical clearing and after photoclearing. Autofluorescence levels were significantly reduced. Scale bars, 500 µm. (C) Quantification of autofluorescence levels after chemical clearing and photoclearing. Values represent average pixel intensity in arbitrary units (A.U.) over ten orthogonal views through the width of the organs. (D) Kidney 1cm-thick section before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (E) Imaging of autofluorescence levels after chemical clearing (upper rows) and photo clearing (lower rows). Scale bars, 500 µm. (F) Quantification of autofluorescence levels after chemical clearing and after photo clearing. Values represent average pixel intensity in arbitrary units (A.U.) over ten orthogonal views through the width of the organs. (G) Lung 1cm-thick section before clearing, after chemical clearing and after photoclearing. Grid scale 1cm. (H) Imaging of autofluorescence levels after chemical clearing (upper rows) and after photo clearing (lower rows). Scale bars, 500 µm. (I) Quantifications of autofluorescence levels after chemical clearing and after photo clearing. Average pixel intensity (A.U.). (J) Common carotid artery segment before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (K) Imaging of autofluorescence levels after chemical clearing (upper rows) and after photo clearing (lower rows). Scale bars, 500 µm. (L) Quantification of autofluorescence levels after chemical clearing and photo clearing. Values represent average pixel intensity in arbitrary units (A.U.) over ten orthogonal views through the width of the organs.

Deep staining of human organs.

(A) Archival thyroid gland lobe before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (B) Maximum intensity projection, planes extracted from a 3D stack of an archival thyroid sample stained with TO-PRO-3 and lectin. Scale bar, 100 µm. (C) 3D volume reconstruction of the archival thyroid sample with merged labeling for TO-PRO-3 and Lectin. Scale bar, 500 µm. (D) Archival esophagus 1cm-thick section before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (E) Maximum intensity projection from a 3D stack subset and (F) 3D volume reconstruction of an archival esophagus tissue stained with TO-PRO-3 and lectin. Esophageal glands are indicated by arrows. Scale bar, 100 µm. (G) Entire ovary before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (H) Sagittal and frontal planes allow the visualization of the inner structure of the ovary. Scale bar, 2 mm. (I) 3D volume segmentation of the ovary and its blood vessels at the level of mesovarium. Color scale volume: purple=0.1mm3 to red=0.7 mm3. Scale bar, 1.5 mm. (J) 3D Quantification of vessels’ straightness in the ovary. Scale bar, 1 mm. (K) Entire testicle before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (L) Maximum intensity projection from a 3D stack subset of a testicle and epididymis labelled with collagen staining. Scale bar, 2 mm. (M) Inset showing the body of the epididymis and its tubules at higher magnification. Scale bar, 200 µm.

Deep immunolabeling of human organs.

(A) 3D volume reconstruction of a kidney section with nephron tubules immunolabeled with aquaporin 2 and vessels labeled with lectin. Scale bar, 1.5 mm. (B) Single plane image extracted from a 3D stack of a kidney section labeled for nephron tubules marker Aquaporin 2 and nuclear stain TO-PRO-3. Glomeruli could be identified with the nuclear staining (arrow). Scale bar, 100 µm. (C) Single plane image extracted from a 3D stack of SMA immunolabeling in the lung. Scale bar, 1 mm. (D) 3D volume reconstruction of SMA labeling in the lung. Scale bar, 1 mm. (E) Penis glans (upper panel) and penis 1-cm thick coronal section (lower panel) before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (F) 3D volume reconstruction of a segment of the panel (I) coronal penis section. Tunica albuginea and cavernous bodies are labeled for collagen with Fast Green. Scale bar, 500 µm. (G) 3D volume reconstruction of a coronal section of the penis. Blood vessels of the cavernous and spongious bodies were immunolabeled for SMA. The urethra could be visualized (arrow). Scale bar, 2 cm. (H) Colon transverse section before clearing, after chemical clearing and after photoclearing. Grid scale 1 cm. (I) 3D volume reconstruction of a human colon sample immunolabeled for SMA and PGP 9.5. The side of the mucosae is indicated by an arrow. Scale bar, 800 µm. (J) Inset from panel (I) with higher-magnification image of the sub-mucosal plexuses. Scale bars, 100 µm. (K) Inset from panel (I) with higher-magnification image of the myenteric plexuses. Scale bars, 400 µm. (L) 3D volume reconstruction the mucosal innervation around colonic crypts (arrows). Scale bar, 70 µm. (M) Inset showing details of a submucosal plexus. Scale bar, 30 µm.

Multimodal alignment of the inner ear.

(A) 3D volume reconstruction of the petrous bone with photogrammetry. Scale bar, 2 mm. (B) Surface rendering of a MRI T2-acquisition of the petrous bone (left panel) and 3D reconstruction of the same volume (right panel). Scale bars, 2 mm. (C) Petrous bone after photoclearing. Grid scale 1cm. (D) 3D volume reconstruction of the cleared petrous bone revealing the intrapetrous path of the facial nerve (arrowhead) and the cochlea (arrow). Scale bar, 2 mm. (E) Overlaid images of the realigned MRI and light sheet microscopy volumes. Scale bar, 2 mm. Right panel shows a magnified image of the inset indicated on the left panel, Scale bar, 1.5 mm. (F) Overlaid single plane image from the realigned MRI and light-sheet microscopy volumes. Scale bar, 1 mm. (G) 3D volume reconstructions of the cochlea viewed from the side. The cochlear nerve innervating the cochlea is visible (arrow). Scale bar, 500 µm. (H) 3D volume reconstructions of the cochlea viewed from above. Scale bar, 500 µm. (I) Single planes images from a 3D acquisition of the cochlea labeled for beta-III tubulin. Left panel shows the facial nerve passing through the petrous bone. Middle panel shows the cochlea (white arrow) divided into the scala vestibuli, media and tympani, together with the utricle (white arrowhead) and a semicircular canal (yellow arrowhead). Right panel shows the ampoullae (yellow arrow). Scale bars, 2 mm. (J) Higher-magnification image of the cochlear membranes and cavities, also showing the organ of Corti (white arrowhead) and spiral ganglions (yellow arrowhead). Scale bar, 500 µm.

mCleLight clears musculoskeletal tissues.

(A) Ventral (left panel) and lateral (right panel) views of a human finger. Left images represent single planes T2-MRI acquisitions and right images show corresponding 3D volume segmentation of the main anatomical structures, including the skin, bone and the flexor tendon (arrow). Scale bar, 1 cm. (B) Left ring finger at successive stages of the clearing process: before clearing, after chemical clearing and after photoclearing. Grid scale, 1 cm. (C) Longitudinal optical section through the finger from the inset in panel B. Scale bars, 2000 µm. (D) Higher-magnification view of individual Pacinian corpuscles of the inset in panel C. Pacinian corpuscles were outlined and observed at various depths, including in the flexor tendon and adjacent to phalangeal bones. The inner core was immunolabeled with S100 antibody. Scale bars, 200 µm. (E) Longitudinal optical section through the finger from the inset in panel B. Scale bars, 2000 µm. (F) Higher-magnification view of an individual Pacinian corpuscle of the inset in panel E. Pacinian corpuscles were outlined and the inner core was immunolabeled with S100 antibody. Scale bars, 200 µm. (G) Dorsal view of a 3D volume reconstruction of a human ring finger with SMA immunolabeling and Fast Green collagen staining. Scale bar, 2 mm. (H) Inset showing higher magnification image of the Fast Green collagen staining outlining the trabecular structure of the phalanx. Scale bar, 700 µm. (I) Lateral view of a 3D volume reconstruction of a human ring finger with SMA immunolabeling and Fast Green collagen staining. Scale bar, 2 mm. (J) Inset showing higher magnification image of the Fast Green collagen staining outlining the fingertip’s reticular collagen network. Scale bars, 1 mm.

Specimen data

Organ-specific decalcification time and light exposure time

Antibodies and dyes used with mCleLight