Three-dimensional hard and soft tissue imaging of the human cochlea by scanning laser optical tomography (SLOT)

Abstract

The present study focuses on the application of scanning laser optical tomography (SLOT) for visualization of anatomical structures inside the human cochlea ex vivo. SLOT is a laser-based highly efficient microscopy technique which allows for tomographic imaging of the internal structure of transparent specimens. Thus, in the field of otology this technique is best convenient for an ex vivo study of the inner ear anatomy. For this purpose, the preparation before imaging comprises decalcification, dehydration as well as optical clearing of the cochlea samples in toto. Here, we demonstrate results of SLOT imaging visualizing hard and soft tissue structures with an optical resolution of down to 15 μm using extinction and autofluorescence as contrast mechanisms. Furthermore, the internal structure can be analyzed nondestructively and quantitatively in detail by sectioning of the three-dimensional datasets. The method of X-ray Micro Computed Tomography (μCT) has been previously applied to explanted cochlea and is solely based on absorption contrast. An advantage of SLOT is that it uses visible light for image formation and thus provides a variety of contrast mechanisms known from other light microscopy techniques, such as fluorescence or scattering. We show that SLOT data is consistent with μCT anatomical data and provides additional information by using fluorescence. We demonstrate that SLOT is applicable for cochlea with metallic cochlear implants (CI) that would lead to significant artifacts in μCT imaging. In conclusion, the present study demonstrates the capability of SLOT for resolution visualization of cleared human cochleae ex vivo using multiple contrast mechanisms and lays the foundation for a broad variety of additional studies.

Advances in Ex Situ Tissue Optical Clearing

“…Notably, it is also beneficial to couple TOC with other than confocal/LSFM imaging techniques, such as optical computed tomography (optical‐CT), optical emission computed tomography (optical‐ECT) and scanning laser optical tomography (SLOT) . These modalities might be perceived as an equivalent of CT which uses visible light, instead of X‐ray, during the imaging, with SLOT characterized by less “stray” light, that is, the light that does not follow primary ray path through the optical system .…”

“…Thus, optical‐CT was successfully applied to evaluate blood vessels of cleared and stained with light‐absorbing inks tissues, such as tumor, heart, or lung . Prominently, optical‐ECT and SLOT detects fluorescent labeling, which means that when applied to well cleared samples these might give both CT‐like and LSFM‐like images almost simultaneously …”

Applicability of vital staining and tissue clearing to vascular anatomy and melanocytes' evaluation of temporal bone in six laboratory species

“…Most morphological analyses use 2‐dimensional images created through histological methods, even though 3‐dimensional techniques exist. For examples, micro CT, scanning Thin‐Sheet Laser Imaging Microscopy, optical micro‐angiography, optical coherence tomography and scanning laser optical tomography have all been tried as 3‐dimensional tools to understand the anatomy and demonstrate the relationship among the various structures (Buytaert, Johnson, Dierick, Salih, & Santi, ; Choudhury, Chen, Shi, Nuttall, & Wang, ; Lee et al, ; Lee, Park, Kang, & Choung, ; Tinne et al, ; Zou et al, ). Unfortunately, these, computer‐assisted reconstructed images have provided limited information when trying to study the in vivo anatomy.…”

“…Tissue‐clearing techniques have been used by many institutions for research in the otologic field. As an example, cochlea analysis with tissue‐clearing technique has been performed in preparation for scanning laser optical tomography (Tinne et al, ), confocal microscopy (MacDonald & Rubel, ), etc. In the present study, we applied tissue‐clearing techniques to create a series of comprehensive and anatomically accurate temporal bone images, without tissue section.…”

Investigation of second harmonic generation and multispectral imaging as new contrast mechanisms in scanning laser optical tomography

“…To further increase the collection efficiency, SLOT was developed in 2011 16 . SLOT uses absorption, scattering and fluorescence as contrast mechanism, that can be provided by the sample intrinsically 17,18 or using a specific labeling 19 . Also the surface of non-transparent samples can be visualized in 3D 20 .…”