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Updated: Dec 26, 2025

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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Published on: December 9, 2013

Spatial filter and its application in three-dimensional single molecule localization microscopy.

Xiaoming Fan¹, Johnny Hendriks, Maddalena Comini

¹Institute of Complex Systems (ICS-4, Cellular Biophysics), Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Institute of Complex Systems (ICS-5, Molecular Biophysics), Forschungszentrum Jülich, Leo-Brandt-Str., 52428 Jülich, Germany. Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 201203 Shanghai, People's Republic of China.

Methods and Applications in Fluorescence

|March 10, 2020

Summary

This summary is machine-generated.

Spatial filter SMLM (SFSMLM) enables 3D imaging by precisely determining axial positions of molecules. This advanced microscopy technique offers nanometer-scale localization for cellular structures.

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Area of Science:

Biophysics
Optical Microscopy
Nanotechnology

Background:

Single molecule localization microscopy (SMLM) achieves super-resolution imaging below the diffraction limit.
Traditional SMLM is primarily a 2D technique, lacking precise axial localization.
Determining the axial position requires supplementary information not inherent in standard SMLM.

Purpose of the Study:

To introduce a novel method, spatial filter SMLM (SFSMLM), for determining the axial positions of fluorescent emitters.
To achieve nanometer-scale axial localization accuracy for molecules and nanoparticles.
To enable three-dimensional imaging of sub-optical resolution cellular structures.

Main Methods:

Utilized two spatial filters placed in separate emission detection channels.
Developed SFSMLM to extract axial position information from fluorescence emission.
Applied the technique to image nanoscale cellular components.

Main Results:

SFSMLM accurately determines axial positions of fluorescent molecules and nanoparticles.
Achieved axial localization with a precision of 15-30 nm over a range of approximately 1.5 μm.
Successfully imaged 3D structures like actin filaments, vesicles, and mitochondria.

Conclusions:

SFSMLM significantly enhances SMLM capabilities by providing accurate axial localization.
The technique allows for high-resolution three-dimensional imaging of cellular ultrastructures.
SFSMLM represents a valuable advancement for nanoscale biological imaging.