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Super-resolution Fluorescence Microscopy

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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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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: Apr 3, 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

Simultaneous two color image capture for sub-diffraction localization fluorescence microscopy.

Ben J Glasgow¹, Lie Ma¹

¹Departments of Ophthalmology, Pathology and Laboratory Medicine, Jules Stein Eye Institute, University of California, Los Angeles, 100 Stein Plaza Rm. BH 623, Los Angeles, CA 90095, USA.

Micron (Oxford, England : 1993)

|September 27, 2015

Summary

This summary is machine-generated.

This study presents a simple fluorescence microscope capable of resolving quantum dots as close as 10nm apart. The technique uses a standard camera and Fourier analysis for precise localization on a flat surface.

Keywords:

CCD camera Diffraction unlimited Fluorescence Localization microscopy Quantum dots Super-resolution microscopy

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

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

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Area of Science:

Optical Microscopy
Nanotechnology
Quantum Dot Imaging

Background:

Accurate localization of nanoscale objects is crucial in various scientific fields.
Existing super-resolution microscopy techniques can be complex and expensive.

Purpose of the Study:

To develop a simple and accessible method for sub-diffraction limit fluorescence localization.
To achieve high-resolution imaging of closely spaced fluorophores on a flat surface.

Main Methods:

Construction of a sub-diffraction limit fluorescence microscope using a standard CCD camera.
Analysis of overlapping Airy discs and point spread functions via Fourier transformation.
Noise reduction and determination of fluorophore coordinates from serial images.

Main Results:

Successfully resolved paired quantum dots with emissions at 540 and 630 nm.
Achieved x-y coordinate resolution of quantum dots separated by as little as 10 nm.
Demonstrated statistical validation for discriminating inter-fluorophore distances with 10 nm variations.

Conclusions:

The developed method offers a simple approach for high-resolution localization of dilute fluorophores on flat surfaces.
This technique is suitable for applications requiring precise spatial determination of nanoscale emitters.
The method is optimized for x-y plane resolution and does not perform serial z-sectioning.