How to orient samples for optimal Stochastic Electrotransport clearing

Stochastic electrotransport (SE) is a novel method for enhancing the transport of electromobile molecules.  SE clearing utilizes rotational electrical field to achieve high speed clearing of biological tissues without tissue damage. Clearing or clarification refers to the removal of lipids from tissues samples in order to improve its optical transparency for imaging and chemical permeability for labeling.

Lipid removal is achieved by the use of surfactant micelles, and the clearing speed of a biological tissue is dependent on how fast the surfactant micelles can travel through the sample. These surfactant molecules are electrically charged and applying electric field accelerates the transport of the surfactant-lipid micelles complexes, enabling much faster clearing.

The SE clearing adjusts parameters such as the strength of the electric field to improve the effective diffusivity of detergent micelles and thereby significantly reduce the total diffusion time compared to that of a simple passive clearing scheme.

Total diffusion time scale (T) is dependent on the diffusion length (l) and the diffusivity of the micelles (D^eff)

However, even with improved diffusivity, the clearing time’s quadratic dependence on the diffusion length remains. Thicker tissues will always take much longer to clear with the diffusion-based methodology. For SE clearing, electric field mediated diffusion occurs mostly along the direction of the electric field. This means that when clearing non-spherical samples, the orientation of the tissue samples undergoing SE clearing can have a significant impact on the total clearing time. As such, utmost consideration should be given to minimize the diffusion length that surfactant micelles will travel when loading the samples into the machine. Consider the following diagram:

The diagram shows two different orientations of a typical mouse hemisphere sample in a SE clearing chamber. The maximum diffusion length along the direction of electrical field for the left sample is almost 3 times longer than that of the right sample. Even with a same sample, the maximum diffusion length is 3 fold different. Because of the aforementioned quadratic dependence on diffusion length, the right sample can take 9 times or even longer to fully clear. Given that the time it takes to fully clear a correctly positioned mouse hemisphere can be up to 3 days, improperly positioned sample can remain uncleared for weeks.  

Optimal sample orientation will allow the tissues to be cleared quickly and without damage. Such orientations can be achieved by using nylon meshes that can help hold the samples in place.