Clearing: A processing technique that renders tissues optically transparent for histology analysis purpose.

Introducing a revolutionary powerful tool for 3D histology of large human ocular samples, including entire eyes, which will be useful in both anatomopathology and in research.

The technology is called “Clearing” and it is a tissue processing technique that removes light-obstructing lipids to render tissues optically transparent. The recent development of light sheet fluorescence microscopy (LSFM) allows volumetric imaging of immunolabeled cleared samples and offers shorter acquisition time than confocal microscopy. It also enables a more comprehensive analysis of relatively large and spatially complex structures, and much faster than complete reconstruction through traditional histology using tissue sections. To date, many clearing protocols for clearing and labeling have been reported. The most common techniques are CLARITY, BABB, 3DISCO, and CUBIC protocols.

Histopathology of an entire eye, in which neuronal, vascular, glial, pigmented, mesenchymal, and immune cells are intimately entangled between a collagenous shield and the vitreous gel, is notoriously difficult because of its heterogeneous composition. Also, because the eye is spherical, 3D reconstructions from parallel sections or from flat mounts are cumbersome and prone to artifacts. Therefore, clearing would offer an elegant solution for histology of intact eyes.

Here we report the achievement of light sheet fluorescence microscopy (LSFM) of entire immunolabelled human eyes by combining a modified iDISCO + clearing protocol, refined procedures for bleaching, and improved antibody penetration using linearization. Acquisition of images through a custom-built extensive numerical dissection enabled multiscale, customized observation of the tissues forming the eye, including pseudo-fundoscopic (i.e., en face) viewing.

Here is the outcome of an eye who underwent clearing and LSFM (Schematic in Fig. 1a). Figure 1c shows a macroscopic view of a human eye after clearing in the imaging cuvette. Figure 1d–k shows the imaging results from a cleared eye of an 84-year-old woman (a schematic of the structures of the human eye is shown in Fig. 1b). The fluorescence signal, labeling ColIV and SMA, showed homogeneous penetration of antibodies and light throughout the sample (Fig. 1d,e). Virtual dissection allowed isolation of the various ocular structures. The entire vascularization of the retina could be visualized similarly to en face clinical imaging (“pseudo-fundoscopy”; Fig. 1f and h). The complex vascular organization of the choroid could be observed in detail in the samples (Fig. 1g). The foveal avascular zone (Fig. 1i) and the multilayered retinal and choroidal circulations (Fig. 1j–k) were clearly defined.

This imaging was performed at The Clinical Investigation Center of the 15-20 National Hospital in Paris, France.

The credit for this article and research goes to:

Darche, M., Borella, Y., Verschueren, A. et al. Light sheet fluorescence microscopy of cleared human eyes. Commun Biol 6, 1025 (2023).

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