Abadie S, Jardet C, Colombelli J, Chaput B, David A, Grolleau J-L, Bedos P, Lobjois V, Descargues P, Rouquette J
Syntivia, Toulouse, France.
ITAV, Université de Toulouse, CNRS, Toulouse, France.
Skin Res Technol. 2018 May;24(2):294-303. doi: 10.1111/srt.12429. Epub 2018 Jan 27.
Human skin is composed of the superimposition of tissue layers of various thicknesses and components. Histological staining of skin sections is the benchmark approach to analyse the organization and integrity of human skin biopsies; however, this approach does not allow 3D tissue visualization. Alternatively, confocal or two-photon microscopy is an effective approach to perform fluorescent-based 3D imaging. However, owing to light scattering, these methods display limited light penetration in depth. The objectives of this study were therefore to combine optical clearing and light-sheet fluorescence microscopy (LSFM) to perform in-depth optical sectioning of 5 mm-thick human skin biopsies and generate 3D images of entire human skin biopsies.
A benzyl alcohol and benzyl benzoate solution was used to successfully optically clear entire formalin fixed human skin biopsies, making them transparent. In-depth optical sectioning was performed with LSFM on the basis of tissue-autofluorescence observations. 3D image analysis of optical sections generated with LSFM was performed by using the Amira software.
This new approach allowed us to observe in situ the different layers and compartments of human skin, such as the stratum corneum, the dermis and epidermal appendages. With this approach, we easily performed 3D reconstruction to visualise an entire human skin biopsy. Finally, we demonstrated that this method is useful to visualise and quantify histological anomalies, such as epidermal hyperplasia.
The combination of optical clearing and LSFM has new applications in dermatology and dermatological research by allowing 3D visualization and analysis of whole human skin biopsies.
人体皮肤由不同厚度和成分的组织层叠加而成。皮肤切片的组织学染色是分析人体皮肤活检组织的结构和完整性的基准方法；然而，这种方法无法实现三维组织可视化。另外，共聚焦或双光子显微镜是进行基于荧光的三维成像的有效方法。然而，由于光散射，这些方法在深度上的光穿透有限。因此，本研究的目的是将光学透明化与光片荧光显微镜（LSFM）相结合，对5毫米厚的人体皮肤活检组织进行深度光学切片，并生成整个人体皮肤活检组织的三维图像。
使用苯甲醇和苯甲酸苄酯溶液成功地对整个福尔马林固定的人体皮肤活检组织进行光学透明化处理，使其变得透明。基于组织自发荧光观察，用LSFM进行深度光学切片。使用Amira软件对LSFM生成的光学切片进行三维图像分析。
这种新方法使我们能够原位观察人体皮肤的不同层和隔室，如角质层、真皮和表皮附属器。通过这种方法，我们轻松地进行了三维重建，以可视化整个人体皮肤活检组织。最后，我们证明了这种方法对于可视化和量化组织学异常，如表皮增生是有用的。
光学透明化与LSFM的结合通过实现对整个人体皮肤活检组织的三维可视化和分析，在皮肤病学和皮肤病学研究中有新的应用。
