biostudies-literatureUnknownXu LWQEuropean Research CouncilNIGMS NIH HHSMobile biological particles, ranging from biomolecules to viral capsids, often diffuse faster than 1<i>μ</i>m²/s, resulting in severe motion blur in conventional millisecond-scale imaging. While shorter exposures may help provide the data needed to capture faster dynamics, quantization of signal intensity per pixel at such exposures eventually interferes with our ability to track. In the extreme case of binary (1-bit-per-pixel) output-where going from 8-bit conventional grayscale imaging to 1-bit directly corresponds to a 255-fold faster acquisition rate-no existing tracking methods can be used, as these methods fundamentally rely on intensity-based localization, which does not leverage the binary output. For this reason, we introduce single-photon single-particle tracking (SP<sbioRxiv : the preprint server for biology2025.01.10.632389https://www.ebi.ac.uk/biostudies/studies/S-EPMC12632838biostudies-literatureSingle-Photon Single-Particle Tracking.PMC12632838R35 GM148237101020445Mitsioni MFSachl RBrossy THof MStastny DXu LWQPresse SRadenovic ARonceray NfalseSingle-Photon Single-Particle Tracking.Mobile biological particles, ranging from biomolecules to viral capsids, often diffuse faster than 1<i>μ</i>m²/s, resulting in severe motion blur in conventional millisecond-scale imaging. While shorter exposures may help provide the data needed to capture faster dynamics, quantization of signal intensity per pixel at such exposures eventually interferes with our ability to track. In the extreme case of binary (1-bit-per-pixel) output-where going from 8-bit conventional grayscale imaging to 1-bit directly corresponds to a 255-fold faster acquisition rate-no existing tracking methods can be used, as these methods fundamentally rely on intensity-based localization, which does not leverage the binary output. For this reason, we introduce single-photon single-particle tracking (SP<s2025-01-01T00:00:00Z2025 Oct2026-06-11T03:14:10.678Z2026-06-11T03:08:25.163ZS-EPMC126328384127948210.1101/2025.01.10.632389