1：Experimental Design and Method Selection

The study involved collecting single-molecule kinetics data of isolated Alexa Fluor 647 molecules on bare and functionalized glass and ITO surfaces. A mathematical model accounting for two reversible dark states was developed to extract rate constants and fractions from the experimental data.

2：Sample Selection and Data Sources

Glass and ITO surfaces were functionalized with alkyl silane or alkyl phosphonic acid, respectively, with varying lengths of the linking alkyl chain. BSA-Alexa Fluor 647 was absorbed onto these surfaces and imaged using dSTORM microscopy.

3：List of Experimental Equipment and Materials

A TILL Photonics iMic TIRF system was used for imaging, including a 640 nm diode laser for illumination, a quad-band dichroic, a 100× Plan-apochromat 1.46 NA oil immersion objective, and an Andor iXon 897 EMCCD camera. Samples were imaged in a magnetic sample chamber under oxygen-scavenging STORM buffer.

4：Experimental Procedures and Operational Workflow

For each imaged region of interest, the active fluorophores were driven into a dark state with high excitation laser power for 1 min. Samples were then immediately imaged for 2000 frames at 100 ms exposure time with reduced laser power. Collected image stacks were saved as raw bytes with an associated Open Microscopy Environment compatible metadata file.

5：Data Analysis Methods

Custom scripts written in MATLAB performed the post-acquisition data analysis, including localization of spots in each image frame, watershed segmentation to identify regions each containing a single active fluorophore, and extraction of photophysical dynamics from segmented trajectories.