Imaging Whole Rodent Brains with Tissue Clearing

Over the last few years we have heard from hundreds of researchers that have said that they have tried tissue clearing and “it did not work.” While this feedback is common from researchers, we have learned that these failures are typically due to a couple of common misconceptions about tissue clearing. To dispel these myths, we will start off by saying that all tissue clearing techniques work, but each technique has its own specific advantages, disadvantages and special considerations. Additionally, some tissue clearing techniques are more challenging than others and slight deviations on the protocol can result in poor results.

Most researchers who try tissue clearing read the original CLARITY paper in 2013 or have seen the recent uDISCO paper on whole mouse tissue clearing. In these publications very large pieces of tissue are cleared and imaged intact in 3D. While these images and videos are incredibly impressive, one must ask themselves if they really need to process and image tissues this large for their specific research question. The reason why this is so important to consider is that imaging tissues deeper than 2 mm dramatically increases the complexity of imaging as well as the time required for tissue processing. Imaging past 2 mm into tissues requires the use of expensive dipping objectives and/or a light sheet microscope.

From our experience, the most common error that researchers make is that they try to replicate these whole tissue imaging protocols right away before understanding the fundamentals of 3D microscopy, tissue labeling or tissue clearing. This often leads a researcher into a situation where they achieve poor results and do not know how to trouble shoot their problem.

Why are we imaging brains in their entirety anyway?

One of the things we commonly forget in our pursuit of cutting edge research is that the original research question we set out for. For example, if our goal is to map the vasculature in an entire mouse brain and better understand tortuosity, why do we need to image the mouse brain in 3D as one whole piece instead of a few large pieces? The reason why this question is very important is that labeling and clearing a whole mouse brain is very expensive, time consuming and challenging. If instead we were to split the mouse brain into a few smaller pieces, we would exponentially decrease the amount of time required to process the tissue for labeling as well as clearing. The pieces could be digitally reconstructed into one tissue later. Additionally, reducing the individual piece sizes to less than 2 mm in thickness would allow the brain to be imaged on just about any 3D microscopy system where whole brains require specialized optics and/or uncommon light sheet microscopy.

To adopt tissue clearing into your workflow, we suggest starting small and beginning with thin sections of tissue and working your way up sequentially as you demonstrate uniform labeling in each thickness of tissue. It is uncommon for your research question to require tissue pieces larger than 2 mm in thickness and we always suggest minimizing overall tissue thickness as it will reduce overall time and cost. The other important consideration to keep in mind is that the current literature on tissue clearing typically describe each clearing technique for a specific application. Protocols for each technique will vary greatly by tissue type and thus it is very important to understand the principles of each technique and how to optimize a protocol for your specific research question. With our Visikol HISTO tissue clearing technique we have embodied this protocol customization in our Protocol Guidebook.

Michael Johnson