Improved clearing of lipid droplet-rich tissues for three-dimensional structural elucidation.

Tissue clearing techniques [1], especially the recently developed CLARITY [2], have enabled three-dimensional imaging of whole tissues and organs by transforming thick tissues into optically transparent and macromolecule permeable gel-like assemblies through the effective removal of lipids without significant alteration to their native structure. The simplicity, convenience, and effectiveness of such methods have made this approach the method of choice in structural elucidations of various tissues and organs that could not be well examined in their native state in toto [3,4]. However, certain tissues remain challenging and are more resistant to clearing procedures, such as fat tissues and those from the liver or muscle that contain large amount of lipid droplets [5,6]. These droplets, which contain a highly hydrophobic and dense core surrounded by a monolayer of phospholipids, range in size from several tens of nanometers in most cells to 100 μm in adipocytes [5]. The hydrophobic core is composed mainly of triglycerides and sterol esters [6], much different from the amphiphilic phospholipids in lipid bilayers that can be easily dissolved by sodium dodecyl sulfate (SDS) or Triton X-100 used in the typical clearing procedure. In fact, it is well known that the extremely hydrophobic triglycerides are difficult to remove by detergents [6]. These dense lipid droplets have a greater index of refraction than their surrounding components and this mismatch can significantly increase light scattering, leading to a noticeable decrease in tissue clarity. Such incomplete clearing severely limits the depth of imaging and achievable resolution even with the most advanced optical systems [7]. There are many important tissues that are highly loaded with such lipid droplets for their functioning, such as adipose tissue, skeletal muscle, liver, and kidney. Arguably, acquiring high resolution three-dimensional information from these ubiquitous and important tissues is essential for our understanding of their basic biology and pathology at the tissue or organ level. To render these lipid dropletrich tissues better cleared, we have developed a simple method, augmenting the existing clearing procedures with an additional process in which the lipid droplets are broken down by pancreatic lipase which is a widely used lipase for hydrolyzing triglyceride. With this easily applied step, we showed that the optical imaging depth of a tissue can be increased by a factor of 5, enabling deeper 3D imaging of these lipid droplet-rich tissues. The detailed protocol of our procedure is described as follows, using mouse liver tissue as a test sample. Following the standard SDSbased passive clearing procedure [2], the intact mouse liver was first fixed by embedding the tissue into a cross-linking hydrogel matrix. After the hydrogel was fully polymerized, the embedded whole liver was sectioned into 1-mm thick sections using a vibratome (Leica, Heidelberg, Germany) and incubated in a 4% SDS solution at 37°C for 11 days. The SDS solution was replaced every day in order to improve the lipid removal efficiency. This step should be more than sufficient to remove most un-crosslinked phospholipids based on published literature and our own experience [8]. Figure 1 shows photographs of the liver section after incubation in SDS for 0, 7, and 11 days. It is clear that even with the 11-day long clearing, the 1-mm thick liver section remains opaque, which is consistent with previously reported results, indicating that the liver tissue cannot be completely cleared simply by increasing the time of SDS-based delipidation [3,4]. Next, we investigated the feasibility of improving liver clearing by pancreatic lipase digestion of the triglycerides, the main components