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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: Sep 29, 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

Multiphoton single-molecule localization by sequential excitation with light minima.

Luciano A Masullo^1,2,3, Fernando D Stefani^4,5

¹Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2390, C1425FQD Ciudad Autónoma de Buenos Aires, Buenos Aires, Argentina.

Light, Science & Applications

|March 25, 2022

Summary

Sequential excitation microscopy achieves 1-2 nm precision for single fluorescent molecules, enabling true molecular resolution imaging. Expanding this to multi-photon microscopy could further enhance localization precision and imaging depth in biological samples.

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Last Updated: Sep 29, 2025

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Area of Science:

Biophysics
Optical Microscopy
Molecular Imaging

Background:

Single-molecule localization microscopy (SMLM) is crucial for high-resolution biological imaging.
Sequential excitation microscopy offers precise localization with reduced light exposure.

Purpose of the Study:

To explore the potential of multi-photon excitation for enhancing single-molecule localization precision.
To investigate methods for achieving deeper tissue penetration in super-resolution microscopy.

Main Methods:

Utilizing sequential excitation principles for single-molecule localization.
Investigating the application of multi-photon excitation regimes.

Main Results:

Achieved 1-2 nm localization precision with moderate photon counts.
Demonstrated potential for true molecular resolution in imaging and tracking.

Conclusions:

Sequential excitation is a powerful technique for high-precision molecular imaging.
Multi-photon excitation may offer complementary benefits for improved localization and deeper imaging in biological specimens.