Systems and methods for particle tracking using spatiotemporal offset light beams
View Patent ↗Systems and methods for particle tracking using spatiotemporal offset light beams. In exemplary embodiments, the optical systems and methods can be used with conventional two-photon microscopy equipment to perform high speed, high precision, and deep tissue three-dimensional single-particle tracking. Exemplary embodiments can be configured for single-molecule studies of biological diffusion and transport processes.
1. A method of tracking a single particle, the method comprising:
separating an input light beam emitted from a light source into a plurality of excitation light beams that are spatially and temporally offset;
illuminating a single particle with the plurality of light beams that are spatially and temporally offset;
detecting fluorescence emitted from the single particle; and
processing photon histogram data resulting from the illumination of the single particle by the plurality of excitation light beams that are spatially and temporally offset to determine a location of the single particle at specific time, wherein processing photon histogram data comprises:
counting and correlating signal photons to a reference clock;
extracting count values from a photon histogram;
selecting a plurality of windows from the photon histogram;
obtaining a time gate value from an average value for each window of the photon histogram;
applying a fluorescence correction factor to the time gate value to generate a corrected time gate value for each window of the photon histogram;
subtracting dark count values from the corrected time gate value for each window of the photon histogram;
calculating error signals from normalized time window differences;
applying proportional control gain values to the error signals; and
performing geometric transform to switch from point-spread function error vectors to galvanic space error vectors.
2. The method of claim 1 wherein the input light beam emitted from the light source is separated into the plurality of excitation light beams via passive beam splitters.
3. The method of claim 1 wherein:
the plurality of excitation light beams that are spatially and temporally offset comprises a first light beam and a second light beam; and
the first light beam is temporally offset between 1 and 20 nanoseconds from the second light beam.
4. The method of claim 3 wherein:
the plurality of excitation light beams that are spatially and temporally offset comprises a third light beam and a fourth light beam; and
the third light beam is temporally offset between 1 and 20 nanoseconds from the fourth light beam.
5. The method of claim 4 wherein the third excitation beam is temporally offset between 1 and 20 nanoseconds from the second excitation beam.
6. The method of claim 1 wherein:
the plurality of excitation light beams that are spatially and temporally offset comprises a first light beam and a second light beam; and
the first light beam is temporally offset between 1 and 10 nanoseconds from the second light beam.