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Super-resolution Fluorescence Microscopy

Super-resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...

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Updated: Aug 24, 2025

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.

Koen J A Martens¹, Martijn Gobes¹, Emmanouil Archontakis²

¹Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands.

|October 21, 2022

Summary

We developed a low-cost spectrally resolved single-molecule localization microscopy (sSMLM) method. This technique improves emitter density and spectral resolution, enabling better discrimination of fluorophores.

Keywords:

Single-molecule spectroscopy multicolor imaging point accumulation for imaging in nanoscale topography (PAINT)single-molecule Förster resonance energy transfer (smFRET)stochastic optical reconstruction microscopy (STORM)

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Last Updated: Aug 24, 2025

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Published on: December 9, 2013

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

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Published on: September 5, 2019

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Published on: October 28, 2018

Area of Science:

Biophysics
Optical Microscopy
Materials Science

Background:

Single-molecule localization microscopy (SMLM) is crucial for high-resolution imaging in life and material sciences.
Simultaneous spectral acquisition in SMLM (sSMLM) faces challenges including optical complexity, low emitter density, and poor spatio-spectral resolution.

Purpose of the Study:

To present a simplified, cost-effective sSMLM implementation addressing current limitations.
To enhance emitter density and spatio-spectral resolution for improved fluorophore discrimination.

Main Methods:

Utilized a single-component, low-dispersion transmission grating placed near the image plane.
Analyzed the +1st diffraction order using standard single-molecule localization algorithms.
Leveraged the spatial separation between the 0th and 1st diffraction orders for spectral information.

Main Results:

Achieved a 5-fold increase in accessible emitter density compared to conventional sSMLM.
Enabled discrimination between fluorophores with peak emission differences as small as 15 nm.
Demonstrated accurate spectral property determination for individual emitters.

Conclusions:

The presented low-cost sSMLM method overcomes key technical hurdles in spectrally resolved super-resolution microscopy.
This approach offers significant advantages for applications requiring the distinction of spectrally similar fluorophores, even under low photon count conditions.