Tutorial: practical considerations for tissue clearing and imaging

Abstract

Tissue clearing has become a powerful technique for studying anatomy and morphology at scales ranging from entire organisms to subcellular features. With the recent proliferation of tissue-clearing methods and imaging options, it can be challenging to determine the best clearing protocol for a particular tissue and experimental question. The fact that so many clearing protocols exist suggests there is no one-size-fits-all approach to tissue clearing and imaging. Even in cases where a basic level of clearing has been achieved, there are many factors to consider, including signal retention, staining (labeling), uniformity of transparency, image acquisition and analysis. Despite reviews citing features of clearing protocols, it is often unknown a priori whether a protocol will work for a given experiment, and thus some optimization is required by the end user. In addition, the capabilities of available imaging setups often dictate how the sample needs to be prepared. After imaging, careful evaluation of volumetric image data is required for each combination of clearing protocol, tissue type, biological marker, imaging modality and biological question. Rather than providing a direct comparison of the many clearing methods and applications available, in this tutorial we address common pitfalls and provide guidelines for designing, optimizing and imaging in a successful tissue-clearing experiment with a focus on light-sheet fluorescence microscopy (LSFM).

Fig. 1 ∣. Considerations in a tissue-clearing experiment utilizing a light-sheet microscope.

The process of tissue clearing involves many steps and many interacting variables throughout the protocol, from fixation to clearing to imaging to data analysis. Many of these variables come together as the sample is being imaged, and the efficiency of the clearing, labeling and imaging protocols interact to reveal the details of the biological sample.

Fig. 2 ∣. Key interactions between variables in a tissue-clearing experiment requiring trade-offs.

Ribbon connections, arrows and source colors indicate cause-effect relationships between features of a tissue-clearing experiment. Connection size indicates relative strength of the relationship for a generic example application, but this particular graph is neither absolute nor quantitative. Similarly, the features listed are not comprehensive but represent a minimal set of interacting parameters to be considered for most tissue-clearing experiments and highlight the interrelated nature of a clearing and imaging processes. Example: both tissue size and image resolution will have large effects on data size, but changing tissue size will also affect many other experimental variables.

Fig. 3 ∣. Artifacts in cleared tissues imaged on light-sheet microscopes.

Image artifacts can result from poor sample preparation, poor clearing and/or limitations of the imaging system. a, Optimal imaging conditions for a well-cleared sample imaged at high axial and lateral resolution resulting in uniform image quality. Images were acquired in sequential planes in the ‘XY’ orientation. The ‘XZ’ orientation represents a resliced image volume postacquisition. Image pictograms are provided to aid in identifying key image features or artifacts in the real images. Scale bar = 1 mm. Aberrations are shown in the orientation(s) where the aberration is most prominent. b, Insufficient clearing results in decreasing image quality with depth. For single-sided illumination, there is a single quality gradient following the light-sheet penetration from right to left in this image. c, RI mismatches can result in generally blurred image due to loss of focus/contrast or image doubling when multiple illumination angles misalign. d, Poor antibody penetration resulting in a ring-like pattern, with most signal near the periphery. e, AF from blood cells (heme) results in high background and bright blood vessels—especially in cases of poor perfusion. f, Sample movement between images can cause misalignment of channels (pictured) or image tiles. g, Image striping can occur when pigments or other absorbing materials block orthogonal illumination light in the sample and light-sheet destriping methods cannot overcome it. The sample was imaged with single-sided illumination from the left. h, Physical distortions such as broken blood vessels (pictured) or nonuniform size changes are common artifacts for some tissue structures. i, Air bubbles from insufficient degassing or mounting will refract light, causing blurring or shadows in the image plane in the case of light-sheet illumination. The sample was imaged with single-sided illumination from the left. j, When viewing the data in the XZ resliced orientation, bubbles appear as distortions. k, Poor axial resolution results in decreased contrast from inclusion of out-of-focus light in a thick optical section. l, Poor light penetration and increased AF at shorter wavelengths are inherent biophysical properties of the light-sample interaction that can be minimized with proper choice of fluorophores. The sample is bright on both sides from dual-sided illumination. m, Spherical aberrations result when there are imperfections in the optical path and cause a halo effect or edge blurring. n, Tile patterning can result when FOV tiling and there is either nonuniform image quality across the FOV, insufficient overlap or computational tiling artifacts. o, Repeated exposure of a region of tissue to excitation light eventually reduces contrast in that plane (image depicts simulated bleaching). p, When viewing the orthogonal XZ orientation, the bleached plane appears as a line.

Fig. 4 ∣. Fluorescence microscopy imaging system geometries and features of microscopes commonly used for imaging cleared tissue.

a, Conventional point-scanning confocal or multiphoton: point-scanning methods require long acquisition times for large tissues. b, Multiview horizontal light sheet: this geometry allows rotation of the sample for multiview illumination and detection and requires movement of the sample for tiling and z-scanning. c, Ultramicroscopy: this configuration mounts the sample from below, which precludes sample rotation for multiview acquisitions. d, Mesoscopic horizontal light sheet—DIY: mesospim.org presents an oversized version of this configuration, and samples up to 50 × 50 × 100 mm can be accommodated with a large FOV. e, Open-top light sheet: this orientation (along with the inverted V-orientation, diSPIM) allows unconstrained sample size in two dimensions for large, flat samples and high-throughput applications. All system geometries are amenable to various magnifications, resolutions and solvent compatibilities, depending on lens choice.