To ensure the ease of use and stability of the device during sampling, the Single-probe is mounted on a microscope glass slide using standard epoxy adhesive (Part No. 20945, ITW Devcon, Inc., Danvers, MA) (Figure ). A Conductive MicroTight Union (M-539, IDEX Health & Science, LLC) connects the fused silica capillary (ID: 50 μm, OD: 150 μm) to the solvent-providing capillary. A PEEK tubing (F-181 and F-380, IDEX Health & Science, LLC) is used as the sleeve of the fused silica capillary to ensure a tight connection. The ionization voltage is applied to the union instead of the nano-ESI emitter, enabling efficient solvent delivery and ionization. To construct a functioning setup, the Single-probe is combined with other components, including a motorized XYZ-stage (CONEX-MFACC, Newport Corp., Irvine, CA), a manual XYZ-translation stage (Compact Dovetail XYZ Linear Stage, New-port Corp., Irvine, CA), a stereomicroscope (Supereyes T004 Digital Microscope, Shenzhen D&F Co., Ltd., Shenzhen, China), and a flexible connector (MXB-3 h, Siskiyou Corp., Grants Pass, OR), integrated on an optical board (Thorlabs Inc., Newton, NJ, US), which is integrated to mass spectrometers (Thermo LTQ Orbitrap XL mass spectrometer, Thermo Fisher Scientific, Inc., Waltham, MA and ) through an interface (Labview) (Figure ). The Single-probe SCMS technique has been used to characterize cellular metabolites through semi-quantitative analysis, in which ion intensities of metabolites are normalized to the total ion current (TIC) as commonly performed in MS studies. The Single-probe semi-quantitative approaches have been used for uncovering molecular diversity and cellular heterogeneity . This section outlines the progression of semi-quantitative applications of the Single-probe SCMS technique in single-cell metabolomics, highlighting its evolution across diverse biological contexts.