biostudies-literatureMeng XEuropean Research CouncilUK Research and InnovationEngineering and Physical Sciences Research Council3111-3118https://www.ebi.ac.uk/biostudies/studies/S-EPMC8532162biostudies-literatureUnknown8(10)Single particle tracking has found broad applications in the life and physical sciences, enabling the observation and characterization of nano- and microscopic motion. Fluorescence-based approaches are ideally suited for high-background environments, such as tracking lipids or proteins in or on cells, due to superior background rejection. Scattering-based detection is preferable when localization precision and imaging speed are paramount due to the in principle infinite photon budget. Here, we show that micromirror-based total internal reflection dark field microscopy enables background suppression previously only reported for interferometric scattering microscopy, resulting in nanometer localization precision at 6 μs exposure time for 20 nm gold nanoparticles with a 25 × 25 μm² field of view. We demonstrate the capabilities of our implementation by characterizing sub-nanometer deterministic flows of 20 nm gold nanoparticles at liquid-liquid interfaces. Our results approach the optimal combination of background suppression, localization precision, and temporal resolution achievable with pure scattering-based imaging and tracking of nanoparticles at interfaces.ACS photonicsMicromirror Total Internal Reflection Microscopy for High-Performance Single Particle Tracking at Interfaces.PMC8532162819593EP/T03419X/1Thorpe SSonn-Segev ADufresne ERYoung GCole DStyle RWMeng XKukura PSchumacher AfalseMicromirror Total Internal Reflection Microscopy for High-Performance Single Particle Tracking at Interfaces.Single particle tracking has found broad applications in the life and physical sciences, enabling the observation and characterization of nano- and microscopic motion. Fluorescence-based approaches are ideally suited for high-background environments, such as tracking lipids or proteins in or on cells, due to superior background rejection. Scattering-based detection is preferable when localization precision and imaging speed are paramount due to the in principle infinite photon budget. Here, we show that micromirror-based total internal reflection dark field microscopy enables background suppression previously only reported for interferometric scattering microscopy, resulting in nanometer localization precision at 6 μs exposure time for 20 nm gold nanoparticles with a 25 × 25 μm² field of view. We demonstrate the capabilities of our implementation by characterizing sub-nanometer deterministic flows of 20 nm gold nanoparticles at liquid-liquid interfaces. Our results approach the optimal combination of background suppression, localization precision, and temporal resolution achievable with pure scattering-based imaging and tracking of nanoparticles at interfaces.2021-01-01T00:00:00Z2021 Oct2025-04-22T07:53:06.893Z2025-04-05T22:20:37.757ZS-EPMC85321623469290110.1021/acsphotonics.1c01268