NanoSpacer: a confinement-based label-free microscopy platform for particle detection, counting, and sizing from...

Label-free particle analysis across micro- and nanoscale particle systems requires multiple specialized instruments, making it difficult to compare measurements across scales within a single workflow. Here we show the use of NanoSpacer/MicroSpacer, a glass-based confinement platform, for particle detection, relative particle counting, and diffusion-based sizing at the single particle level. The platform confines liquid samples between spacer-defined glass surfaces, restricting the vertical observation volume while allowing particles to move freely within the imaging plane. This improves particle visibility under darkfield illumination and supports more reliable particle tracking for diffusion-based sizing in solutions. Diffusion coefficients are obtained from mean-squared displacement (MSD) analysis using open-source software and converted to hydrodynamic size via the Stokes-Einstein relationship. The platform was evaluated across four model systems spanning micro- to nanoscale: (i) binary GFP/mKate bacterial mixtures were quantified at the single-cell level, with GFP ratios of 59%, 18%, and 89% in agreement with correlative flow cytometry (65%, 23%, 86%) and colony-forming unit counts (54%, 17%, 86%); (ii) bacteriophages Polaria and Mystique were detected by darkfield and apparent size estimates returned modal hydrodynamic diameters of approximately 250 nm and 190 nm, consistent with electron microscopy; (iii) putative nanobubbles were produced by chemical and mechanical methods, with modal diameters of approximately 170 nm and 600 nm; (iv) polystyrene nanoplastic particles (100, 200, and 1000 nm) were imaged in freshwater and seawater, where salt-induced aggregation produced clusters spanning orders of magnitude in size. Together, these results establish NanoSpacer/MicroSpacer as a cost-efficient, microscopy-compatible workflow for particle metrology across micro- and nanoscale systems relevant to marine science and microbiology.