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Related Experiment Video

Updated: Aug 12, 2025

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles
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Multicolor 3D Orbital Tracking.

Frank Mieskes1,2,3, Evelyn Ploetz1,2,3, Fabian Wehnekamp1,2,3

  • 1Department of Chemistry and Center for NanoScience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstraße 11, 81377, Munich, Germany.

Small (Weinheim an Der Bergstrasse, Germany)
|January 29, 2023
PubMed
Summary
This summary is machine-generated.

This study introduces novel dual-color 3D orbital tracking for independently following multiple biomolecules. This advanced single-particle tracking (SPT) method enables simultaneous observation of molecular interactions and dynamics.

Keywords:
Forster resonance energy transfer (FRET)active feedback trackingdual-color trackingreal-time three-dimensional (3D) single particle trackingsingle molecule spectroscopy

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Molecular Biology

Background:

  • Single-particle tracking (SPT) offers high spatiotemporal resolution for studying particle dynamics.
  • Current multi-color SPT methods are limited, with no existing technique capable of independently tracking two distinct biomolecules in four dimensions.
  • Investigating molecular interactions requires advanced tracking capabilities for multiple species.

Purpose of the Study:

  • To demonstrate new modalities for dual-color 3D orbital tracking.
  • To enable independent tracking of multiple, differently labeled biomolecules in four dimensions.
  • To showcase the application of this technique for studying molecular interactions and dynamics.

Main Methods:

  • Development and implementation of dual-color 3D orbital tracking using a second detection channel.
  • Experiments involving independently diffusing particles of different types.
  • Tracking correlated motion of interacting particles by monitoring one species in one channel and the second in another.
  • Utilizing 3D orbital tracking in one channel while monitoring spectral signatures in a second channel for Förster Resonance Energy Transfer (FRET) measurements.

Main Results:

  • Successful demonstration of dual-color tracking for independently diffusing particles.
  • Implementation of a method to track correlated motion of interacting particles.
  • Accurate readout of Förster Resonance Energy Transfer (FRET) values over time for mobile particles using spectral signature analysis.

Conclusions:

  • The developed dual-color 3D orbital tracking significantly expands the capabilities of single-particle tracking.
  • This technique allows for independent, four-dimensional tracking of multiple biomolecules and their interactions.
  • The method provides a powerful tool for quantitative analysis of molecular dynamics and energy transfer in biological systems.