The subseafloor biosphere hosts vast and diverse microbial communities, yet many of its members remain “invisible” because cells are extremely sparse and buried in a geological matrix. This review summarizes methodological advances that enable life detection in low-biomass subseafloor sediments and rocks. We highlight (i) discriminative cell enumeration (SYBR-DiCE), which mitigates non-specific adsorption of DNA dyes to particles; (ii) density-based cell separation and clean handling that increase analytical sensitivity; (iii) single-cell substrate incorporation probing using incubation coupled to NanoSIMS, which revealed high fractions of active and revivable cells in ultraoligotrophic sediments; and (iv) synchrotron micro-CT approaches to visualize pore networks and potential cell distributions, including iodine-bearing ionic liquids with K-edge differential imaging and osmium staining. By integrating quantitative enumeration, single-cell activity, and 3D microstructure, next-generation life detection will better constrain the limits of life and its biogeochemical significance in the deep biosphere.
