(A) Physical isolation can be broadly categorized into three main approaches: laser capture microdissection (LCM) of regions of interest (ROIs) (55), direct aspiration of target cells using a micropipette guided by a micromanipulator (6), and unphotocaging of ROIs through light of a specific wavelength (5). Specifically, the photochemical approach selects ROIs by photocaging and uncaging of photocleavable adapters on the nucleic acid molecule. (B) Spatial barcoding typically constructs a 2D mosaic composed of multiple pixels via microfluidic channels (9) or microbead arrays (82) that each carry a unique DNA barcode. Subsequently, cells and their contents within each pixel are tagged with a unique spatial coordinate. (C) Fluorescence imaging directly visualizes the spatial location of target molecules through in situ fluorescence imaging. (D) MS imaging ionizes molecules in the sample using a laser beam (MALDI) or electrospray (desorption electrospray ionization, DESI). (E) Label-free optical imaging. It captures specific molecules by detecting transmitted light, scattered light, exciting light, or intrinsic emission from cells or tissues, without the need for staining. QPI, quantitative phase imaging; SHG, second harmonic generation; TPEF, two-photon epifluorescence.