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Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

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Self-Referenced Scanning Ion Conductance Microscopy for Bath Solution-Flexible Imaging.

Jian Zhuang^1,2,3, Qiangqiang Zheng^1,2, Shengbo Gu^1,2

¹Key Laboratory of Education Ministry for Modern Design Rotor-Bearing System, Xi'an Jiaotong University, Xi'an 710049, China.

Analytical Chemistry

|September 29, 2025

Summary

A new self-referenced scanning ion conductance microscopy (SR-SICM) allows high-resolution imaging in diverse solvents. This advancement overcomes limitations of conventional SICM, enabling stable imaging in previously inaccessible environments.

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Area of Science:

Nanotechnology
Surface Science
Biophysics

Background:

Scanning ion conductance microscopy (SICM) is vital for in situ imaging in liquids.
Conventional SICM requires identical electrolyte concentrations, limiting its use in asymmetric or electrolyte-free conditions.

Purpose of the Study:

To develop a modified SICM technique for high-resolution imaging in solvent-flexible environments.
To overcome the limitations of conventional SICM in asymmetric or electrolyte-free conditions.

Main Methods:

Developed a single pipette-based self-referenced SICM (SR-SICM) with a silver-coated nanopipette exterior.
Utilized Finite Element Method (FEM) simulations to understand SR-SICM operation and optimize parameters.
Experimentally validated SR-SICM performance with PDMS structures and biological samples.

Main Results:

SR-SICM enables stable, high-resolution imaging in diverse solvent systems, including electrolyte-free conditions.
FEM simulations revealed a nonmonotonic current-distance profile due to ion transport effects.
Experimental results confirmed an extended feedback range compared to conventional SICM.

Conclusions:

SR-SICM significantly broadens the applicability of SICM technology.
This method allows stable imaging in environments previously inaccessible to conventional SICM.
SR-SICM offers a robust and versatile tool for biomedical and materials science research.