In Situ "Confocal" Electrochemiluminescence 3D Imaging: From Cell to Tissue Section

Endowing electrochemiluminescence (ECL) imaging technique with three-dimensional (3D) resolution to investigate specimens at varying axial depths poses a challenging yet significant objective. Herein, a "confocal" 3D ECL imaging method was developed using luminol as ECL probe, in which excited luminophore was formed in the vicinity of electrode surface through homogeneous chemical reactions between oppositely diffusing ECL precursors, luminol diazaquinone intermediate (L), and hydrogen peroxide (H₂O₂), confining the ECL emission in a thin plane (ECL focal plane) parallel to electrode surface at their intersection. The regulating ability of electrochemical method on the reaction fluxes of L and H₂O₂ was validated, regulating the axial location of the ECL focal plane from 0 to 63 µm, which can even extend to 400 µm by using the stable coreactant of ClO^-. Leveraging the optical sectioning capability of the ECL focal plane, the "confocal" 3D ECL imaging method was applied to bioimaging, from cells to tissue sections. It revealed cellular morphology changes during cell polarity establishment and the heterogeneous distribution of complex tubule structure in kidney tissue sections. The optical sectioning capability of "confocal" 3D ECL imaging makes it a powerful tool for studying complex biological samples.