Visualizing and Calculating Tip–Substrate Distance in Nanoscale Scanning Electrochemical Microscopy Using 3-Dimensional Super-Resolution Optical Imaging

Abstract

We report a strategy for the optical determination of tip-substrate distance in nanoscale scanning electrochemical microscopy (SECM) using three-dimensional super-resolution fluorescence imaging. A phase mask is placed in the emission path of our dual SECM/optical microscope, generating a double helix point spread function at the image plane, which allows us to measure the height of emitting objects relative to the focus of the microscope. By exciting both a fluorogenic reaction at the nanoscale electrode tip as well as fluorescent nanoparticles at the substrate, we are able to calculate the tip-substrate distance as the tip approaches the surface with precision better than 25 nm. Attachment of a fluorescent particle to the insulating sheath of the SECM tip extends this technique to nonfluorogenic electrochemical reactions. Correlated electrochemical and optical determination of tip-substrate distance yielded excellent agreement between the two techniques. Not only does super-resolution imaging offer a secondary feedback mechanism for measuring the tip-sample gap during SECM experiments, it also enables facile tip alignment and a strategy for accounting for electrode tilt relative to the substrate.

A snapshot of the electrochemical reaction layer by using 3 dimensionally resolved fluorescence mapping

“…These phenoxazines are indeed becoming increasingly popular in coupled EC fluorescence studies. 18 – 20 The oxidized form RZ is only fairly fluorescent ( λ = 638 nm, φ = 0.11). 21 RF, which is electrogenerated through a two-electron irreversible reduction of RZ, is highly fluorescent ( λ = 592 nm, φ = 0.41).…”

Real‐time Conversion of Electrochemical Currents into Fluorescence Signals Using 8‐Hydroxypyrene‐1,3,6‐trisulfonic Acid (HPTS) and Amplex Red as Fluorogenic Reporters

“…In the literature, it has been shown that such kind of accumulation could be avoided by introducing in solution a species which chemically scavenges the fluorescent molecules produced at the electrode. [30,39] In the case of the HPTS/BQ system, Compton et al showed that the spatial extend of the fluorescence profile developing at an electrode where BQ is reduced could be minimized by acidifying the medium with HCl, since protonation of PTS À into its non-fluorescent HPTS then occurred closer from the electrode surface. [10] We thus explored this pH-based strategy as a way of solving the "fluorescence hysteresis" problem we encountered here.…”

Advances and Perspectives in Chemical Imaging in Cellular Environments Using Electrochemical Methods

“…Methods of tip-substrate distance regulation for SECM [27] include simply using the faradaic current, the use of impedance in alternating current (AC)-SECM [28], the use of oscillating probes in tip-position modulation (TPM)-SECM [29], shear-force SECM [30], intermittent contact (IC)-SECM [31], and three-dimensional super-resolution optical imaging [32]. There are advantages and limitations to each of these techniques, for example the faradaic current is somewhat limited by the fact that the current response at an electrode is affected by both the tip-substrate separation distance and the electroactivity of the underlying substrate.…”