Stochastic Electrotransport of Activity-Modulated Molecules for Rapid and Scalable 3D Phenotyping

For decades, sectioning-based two-dimensional molecular phenotyping techniques have been extensively used for investigation of tissue samples across biology and medicine. These techniques ensure that molecular targets in ultrathin tissue slices experience relatively similar reaction conditions (e.g. probe concentration and reaction time) to achieve complete and uniform labeling of given samples. However, three-dimensional information is lost unless a sophisticated and laborious 3D reconstruction is employed. CLARITY technology has succeeded in proving that an intact, full brain can be labeled with molecular probes, but penetration is poor because passive diffusion of molecular probes in the dense nanoporous mesh is slow (Chung et al., Nature 2013). Even near the surface, labeling is highly non-uniform due to large variations in probe concentration and in the probe-target interaction time across superficial layers. Moreover, labeling of the crucial inner structures remains incomplete even after months. These critical limitations of the current techniques have prevented us from obtaining system-wide quantitative molecular information from large scale intact tissues. Here we introduce a new technology (termed eTANGO) that addresses these challenges by integrating two novel concepts: stochastic electrotransport and dynamic affinity shift. The penetration problem is solved by stochastic electrotransport, which selectively and rapidly drives only charged molecules (e.g. antibodies and RNA probes) without disrupting the surrounding charged matrix (e.g. brain). Furthermore, probe-target binding affinities are dynamically modulated to synchronize reaction times brain-wide. Integration of these two concepts enables all the endogenous molecular targets in billions of cells to experience the same reaction condition (time and concentration). This yields complete and uniform staining of the intact brain within hours. We applied eTANGO to immunostain various cellular, molecular and structural markers a variety of molecular probes, including organic dyes, carbohydrate-binding proteins, and immunoglobulins. The unique strength of eTANGO -- complete homogeneous immunostaining -- opens the possibility of high-throughput brain-wide proteomic profiling at single cell resolution. Together with CLARITY, we anticipate eTANGO to facilitate integrated understanding of large-scale intact biological systems.