3D imaging of cleared ex vivo normal human skin, skin appendages and psoriasiform skin lesion using light-sheet microscopy (LSFM)

USING LIGHT-SHEET FLUORESCENCE MICROSCOPY (LSFM)

Confocal and multiphoton microscopies have emerged as useful imaging tools for the non-invasive visualization of normal and pathological skin with high resolution. However, these techniques are characterized by a limited penetration depth due to light scattering and can cause photobleaching. Light-sheet fluorescence microscopy (LSFM) is an attractive approach that has shown promise for the acquisition of volumetric data of thick tissues. In this study, LSFM was combined with optical clearing methods to allow in-depth optical sectioning and generation of 3D images of entire human skin biopsies.

Skin topical clearing and skin imaging metholody

1. OPTICAL CLEARING OF HUMAN SKIN BIOPSIES

Illustration showing a human skin biopsy before and after optical clearing with ethanol and benzyl alcohol/benzyl benzoate

2. LSFM OF HUMAN SKIN BIOPSIES AND IMAGE PROCESSING

Illustration showing light sheet cross cleared skin and image processing with light sheet fluorescence microscopy

Imaging of a whole human skin biopsy

A 1.7 mm z-stack of a human skin biopsy was imaged with LSFM using an excitation laser of 488 nm and emission filter of 525/50 nm. (A) Slice at 0 μm, (B) 460 μm, (C) 1200 μm and (D) 1400 μm depth is shown. Skin biopsies were obtained from female donors aged between 32-42 years old. Scale bar: 1000 μm.

Four 3D images of skin biopsy with light sheet fluorescence microscopy
Volume rendering of a human skin with Amira® Software
Healthy skin                                 Psioriasis skin lesion
Imaging quantification of epidermal hyperplasia
Imaging and analysis of epidermial hyperplasia in a psoriasiform skin lesion.

Identification of morphological features associated with psoriasis in 3D

H&E staining of (A) healthy skin and (B) pathological skin. Epidermis segmentation and volume rendering of (C) healthy skin and (D) pathological skin following LSFM (excitation laser of 488 nm, emission filter of 525/50 nm). The volume of pathological skin epidermis (0,092 mm3) was almost twice as large as that of healthy skin (0,053 mm3). The pathological skin had a thickness of 203,7 μm compared to 117,9 μm for healthy skin. Psoriasiform skin lesion was obtained from a female donor. Scale bar: 100 μm.

Visualization of human skin appendages entire structure in 3D

Sweat glands (A-C), sebaceous glands (D-F), hair follicle bulbs (G-I) and the cutaneous plexus (J-L) were observed using H&E staining performed on skin cross-sections (for A, D, G and J) or optical sections of autofluorescence skin extracted from z-stacks acquired with Light-Sheet Fluorescence Microscopy (B, E, H and K). Volume reconstructions of human skin appendages were performed with Amira® software (C, F, I and L). (A, B) Arrows and arrowheads point to sweat ducts and conduit holes, respectively. (D, E) Asterisks and arrowheads show the cytoplasm and membranes of sebum-secretory cells, respectively. (G, H) Single asterisks, double asterisks and arrows show dermal papilla, hair shaft and inner root sheath, respectively. (J, K) Asterisks, arrows and arrowheads point to the lumen, smooth muscle and endothelium of blood vessels, respectively. Scale bars 200 μm.

Visualization of human skin appendages entire structure in 3D
3D imaging of human skin appendages.

Conclusion

LSFM combined with optically cleared biopsies enables 3D in-depth visualization of different skin compartments, appendages, and psoriasis lesion both at the macroscopic and cellular levels with high resolution. It opens new perspectives in dermatological research to study normal skin morphology and morphological changes associated with skin pathologies.

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