In neuroscience, and indeed in the broader field of biomedical science, imaging of fixed tissue sections is routine. Researchers are typically limited by the depth of tissue that can be imaged due to light absorption and light scatter (caused largely by lipid content) and thus tissue is frequently sliced into sections up to 100 micrometres.
To circumvent the requirement for thin tissue sections, researchers have developed various methods to homogenise the tissue refraction of light and reduce scatter, collectively known as tissue clearing. The most promising and widely adopted of these methods, CLARITY, removes lipids from the tissue through hydrogel-based tissue clearing. The method is not free from problems however and causes swelling of the tissue which has not, until now, been quantitatively explored satisfactorily.
In their paper, published in Scientific Reports, the authors discuss the need for a reliable protocol for tissue clearing whose effects on tissue morphology have been quantified and validated. Following their protocol, the they quantified the expansion across the tissue and found that the variance introduced was not enough to affect biological conclusions and indeed was generally less than the natural variability associated with uncleared tissue. Furthermore, fixatives such as PFA can shrink brain tissue so it is possible that methods such as CLARITY, associated with tissue expansion, can return the tissue more closely to the measurements found in vivo.
Tyson et al.’s protocol is incredibly useful for the field and beyond. It allows for the reliable investigation of neuroanatomy, offering evidence to support hydrogel-based brain tissue clearing as a valid method to image large volumes of tissue.
Corresponding author, Laura Andreae, commented:
“Optical clearing approaches such as CLARITY are increasingly being used to allow large-scale 3D imaging of cellular structures, especially in brain tissue, but concerns about hydrogel-based tissue expansion affecting cellular morphology remained. Here, we show that in fact such changes are similar across spatial scales, giving reassurance to the field, and also provide what we hope will be useful information about the use of post-clearing labelling of cellular structures with antibodies and small molecule dyes.”