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Related Experiment Video

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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
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Multi-Dimensional Spectral Single Molecule Localization Microscopy.

Corey Butler1,2, G Ezequiel Saraceno1, Adel Kechkar3

  • 1Univ. Bordeaux, CNRS, Interdisciplinary Institute for Neuroscience, IINS, UMR 5297F-33000, F-33000, Bordeaux, France.

Frontiers in Bioinformatics
|October 28, 2022
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Summary
This summary is machine-generated.

This study introduces a new dual-objective spectral imaging method for simultaneous 3D single particle tracking of multiple proteins in living cells. The technique enhances molecular visualization without sacrificing resolution, enabling deeper biological insights.

Keywords:
live cell imagingmulti-emitter fittingsingle molecule localizationsingle particle trackingspectral imaging

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Area of Science:

  • Cellular and Molecular Biology
  • Biophysics
  • Microscopy and Imaging Technologies

Background:

  • Single Particle Tracking (SPT) and Single Molecule Localization (SML) techniques provide nanoscale insights into cellular organization and dynamics.
  • Current SPT methods face limitations in simultaneously tracking multiple proteins, often requiring trade-offs in resolution, field of view, or the number of detectable colors.
  • Simultaneous high-resolution tracking of multiple molecular species within large fields of view is crucial for understanding complex cellular processes.

Purpose of the Study:

  • To develop and validate a novel dual-objective spectral imaging configuration for simultaneous 3D single particle localization and tracking (3D SPT) of multiple distinct molecular species.
  • To overcome the limitations of conventional SPT setups by enabling multi-species tracking without compromising spatio-temporal resolution over large fields of view.
  • To provide a versatile and user-friendly platform for advanced SMLM applications in live cell imaging.

Main Methods:

  • Implementation of a dual-objective spectral imaging setup using commercially available microscopes, incorporating a dispersive element in one optical path to spectrally separate emitters.
  • Development of dedicated computational tools, including the PALMTracer software, for quantitative analysis of 3D + t + λ SMLM data.
  • Validation of the system through multi-color 3D DNA-PAINT on fixed samples and simultaneous tracking of multiple receptors in live fibroblast and neuron cultures.

Main Results:

  • Demonstration of simultaneous 3D single particle localization and tracking of up to five distinct fluorescent species over large fields of view.
  • Achieved high spatio-temporal resolution (<30 nm, millisecond) for multiple tracked species without compromise.
  • Successful application in multi-color 3D DNA-PAINT and live-cell imaging of receptor dynamics in neurons and fibroblasts.

Conclusions:

  • The developed dual-objective spectral imaging system offers a powerful and flexible solution for multi-species 3D SPT in live cells.
  • This approach significantly advances the capability to study complex molecular interactions and dynamics at the nanoscale.
  • The freely available PALMTracer software facilitates the quantitative analysis of advanced SMLM data, promoting broader adoption of the technique.