3DISCO

The method was developed by the team of Ali Ertürk and Hans-Ulrich Dodt at the Max-Planck-Institute in Munich,[1][2] and it was aimed primarily at clearing and imaging unsectioned mouse brain and spinal cord samples.

Later on, the original method or modified versions of it were successfully used in many fields of biological research to image and investigate whole body of mice,[3] structure and function within the mouse brain,[1] stem cells,[4][5] tumor tissues,[6][7][8] developmental processes,[9][10] or whole human embryos.

[19] As result the light is scattered on its path through the tissue, which leads to decrease in resolution or even to disappearance of signal in samples thicker than a few tens of micrometers.

[20][21] After fixation with (usually with paraformaldehyde) and eventually labeling with dyes, the samples are dehydrated via incubation in solutions with growing concentration (50%, 70%, 80% and 100% in water) of tetrahydrofurane (THF).

[1] 3DISCO protocol is best suited for fixed tissues labeled with strong fluorophores, ideally transgenic models expressing fluorescent proteins such as GFP (alternatively staining with synthetic dyes is possible as well).

[1][10] Main drawbacks of this method are partial delipidation of tissue during clearing discriminating use of lipophilic dyes, shrinkage of tissue during clearing,[21] partial degradation of fluorescence,[22] complete degradation of fluorescence during long term storage[1] and toxicity of used reagents (and their potential to damage the microscopy objectives if leak from imaging chamber).

[8] Authors of iDISCO (stands for “immunolabeling-enabled imaging of solvent-cleared organs“) included pretreatment of sample with methanol, hydrogen peroxide, detergents and dimethyl sulfoxide (DMSO) together with antibody labeling before clearing.

[10] Authors of uDISCO (from “ultimate imaging of solvent-cleared organs“) enhance a shrinkage of tissue, a common bystander effect of dehydration of sample in first step of clearing.

[3] It is worth to mention that uDISCO was highlighted by media worldwide including New York Times,[26] Wall Street Journal,[27] Business Insider,[28] Nature and Science[29] magazines.

Authors of 3DISCO method used it first for studying regeneration in the central nervous system (CNS) of mouse, including counting of microglia, astrocytes and mapping trajectories of axons after injury.