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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Published on: June 30, 2018

Time-modulated excitation for enhanced single-molecule localization microscopy.

Pierre Jouchet¹, Christian Poüs², Emmanuel Fort³

¹Université Paris-Saclay, CNRS, Institut des Sciences Moléculaires d'Orsay, 91405 Orsay, France.

Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences

|February 14, 2022

Summary

Structured illumination microscopy (ModLoc) enhances molecular position accuracy using a modulated fluorescence signal. This method improves localization precision by 2.4x, offering a significant advancement for super-resolution imaging.

Keywords:

fluorescence lock-in modulation single-molecule localization microscopy

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Published on: June 30, 2018

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Published on: November 23, 2021

Area of Science:

Biophysics
Optical Microscopy
Nanotechnology

Background:

Single-molecule localization microscopy (SMLM) aims to improve spatial resolution.
Standard SMLM methods face limitations in localization precision.
Structured illumination offers potential for enhanced molecular positioning.

Purpose of the Study:

To develop a novel SMLM technique for improved localization precision.
To investigate the use of time-shifted sinusoidal excitation patterns.
To overcome camera acquisition frequency limitations in SMLM.

Main Methods:

Implemented time-shifted sinusoidal excitation patterns to modulate fluorescence signals.
Developed fast demodulation systems upstream of the camera.
Synchronously sampled temporally modulated fluorescence signals on-camera.

Main Results:

Achieved a 2.4x improvement in localization precision in one direction compared to Gaussian fitting.
Demonstrated the ModLoc microscopy technique experimentally.
Identified kilohertz modulation frequencies as necessary for short-lifetime emitters.

Conclusions:

ModLoc microscopy significantly enhances localization precision in SMLM.
Current camera acquisition rates limit the full potential of ModLoc.
Future development requires demodulation systems capable of kilohertz frequencies.