Since it was first introduced in 2014, the Single-probe-based methods have demonstrated their capabilities in various studies of microscale bioanalyses, such as single cells, tissue slices, and 3D tumor models, in ambient conditions. Implemented with other techniques in instrumentation (e.g., microscopy and precise manipulation), chemical reactions, and surface functionalization, applications of these methods have been largely extended. The advancement in data analysis tools (e.g., multivariate analysis and machine learning) enables extraction of essential information from complex data. Regardless of their advantages, broad applications of the Single-probe-based methods still face multiple challenges. In Single-probe SCMS studies, cell sampling needs to be manually performed using the XYZ-stage system guided by a microscope. Although this is beneficial for studies of target cells, which can be labeled by dyes or fluorescent proteins, among heterogenous populations, these manual procedures largely limit the analysis throughput. In fact, microfluidics techniques have been implemented to SCMS metabolomics studies . Similar strategies can be potentially adopted by the Single-probe SCMS setup to improve its analysis throughput. In Single-probe MSI studies, maintaining the robustness of the experimental setup for stable data acquisition (e.g., several hours) has been challenging. These issues can be mitigated by fabricating robust probes with carefully adjusted tip sizes and shapes. In fact, taking advantage of modern microfabrication techniques (e.g., micromachining, microinjection, and 3D printing), the fabrication of high-quality Single-probe devices can be automated and standardized, promoting their widespread adoption with high consistency and reliability across laboratories. In addition, enclosed, environmentally controlled setups can further enhance reproducibility by mitigating external influences such as temperature and humidity variations. In principle, the Single-probe setup can be customized and coupled with any model of mass spectrometer with a suitable interface. Its open design allows for flexible customization of translation stage system, microscope, and solvent and reagent selection and delivery. Similar to all other MS studies, the Single-probe MS techniques can reap the benefit of rapid advancements in modern mass spectrometers (e.g., improved detection sensitivity, mass resolution, data acquisition speed). Collectively, these