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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 developed.

Three-Dimensional Microscopy in Microbiology

Three-Dimensional Microscopy in Microbiology

Three-dimensional imaging techniques are essential in cell biology, allowing researchers to visualize intricate cellular structures with high resolution. Two prominent methods, Differential Interference Contrast Microscopy (DIC) and Confocal Scanning Laser Microscopy (CSLM), provide distinct advantages for imaging live and thick specimens, respectively.Differential Interference Contrast MicroscopyDIC microscopy enhances contrast in transparent, unstained samples by converting phase...

Confocal Fluorescence Microscopy

Confocal Fluorescence Microscopy

Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...

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Updated: Jun 25, 2026

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

Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point

Sri Rama Prasanna Pavani¹, Michael A Thompson, Julie S Biteen

¹Department of Electrical and Computer Engineering, University of Colorado, Boulder, CO 80309, USA.

Proceedings of the National Academy of Sciences of the United States of America

|February 13, 2009

Summary

This summary is machine-generated.

This study presents a novel wide-field microscope using a double-helix point spread function (DH-PSF) for 3D super-resolution imaging. This technique achieves nanoscale precision, overcoming the optical diffraction limit for advanced molecular visualization.

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Last Updated: Jun 25, 2026

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

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Published on: September 29, 2014

Area of Science:

Optical microscopy
Nanotechnology
Biophysics

Background:

Traditional optical microscopy is limited by the diffraction limit, restricting resolution in 3D imaging.
Super-resolution techniques aim to surpass this limit but often face challenges in speed, precision, and applicability to thick samples.

Purpose of the Study:

To demonstrate 3D single-molecule fluorescence imaging beyond the optical diffraction limit using a wide-field microscope.
To achieve high-precision localization and super-resolution imaging in thick samples.

Main Methods:

Development and implementation of a wide-field microscope featuring a double-helix point spread function (DH-PSF).
Localization of single fluorescent molecules in 3D using the DH-PSF's unique properties (rotating lobes).
Utilizing photoactivatable fluorophores for repeated imaging of sparse subsets to reconstruct high-density molecular distributions.

Main Results:

Achieved 10- to 20-nm localization precision in 3D over a 2-micrometer depth of field in single 500-ms acquisitions.
Demonstrated super-resolution imaging of densely packed molecules in thick polymer samples.
The DH-PSF design showed high and uniform Fisher information, crucial for precise localization.

Conclusions:

The combination of DH-PSF optical design and digital postprocessing enables 3D super-resolution imaging beyond the Rayleigh diffraction limit.
This approach offers a powerful tool for high-resolution visualization in biological and material science applications.
The method provides a pathway to significantly improve 3D imaging resolution and depth penetration.