1：Experimental Design and Method Selection:

The study involves the development and use of a near-field desorption postionization time-of-flight mass spectrometer (NDPI-TOFMS) system, which integrates a nano-aperture tip (nano-AT) ion source, a self-developed TOFMS, and a built-in CCD observation system. The method is based on laser desorption and postionization for mass spectral analysis and atomic force microscopy (AFM) for topographical imaging.

2：Sample Selection and Data Sources:

HeLa cells were used as the biological samples, incubated with compounds such as proflavine and methylthioninium chloride. Data were acquired from mass spectra and AFM images of these cells.

3：List of Experimental Equipment and Materials:

Key equipment includes the NDPI-TOFMS system, nano-AT ion source, tuning fork for AFM, 532 nm laser for desorption, 157 nm laser for postionization, CCD camera, and SEM for tip imaging. Materials include HeLa cells, proflavine, neutral red, methylthioninium chloride, and potentially MALDI matrix.

4：Experimental Procedures and Operational Workflow:

The workflow involves using the nano-AT to desorb material from the cell surface with a laser, followed by postionization and mass spectrometric detection. Topographical information is obtained concurrently via AFM. Specific steps include autofocusing for irregular surfaces, acquiring mass spectra with 2 laser pulses per pixel, and generating images with pixel sizes down to 250 nm.

5：Data Analysis Methods:

Data analysis includes calculating lateral and imaging resolutions from line-scan profiles, determining absolute limit of detection (ALOD) from calibration curves, and correlating chemical and topographical data for 3D reconstruction. Statistical methods such as signal-to-noise ratio (SNR) and standard deviation are used.