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Related Concept Videos

Atomic Force Microscopy01:08

Atomic Force Microscopy

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Atomic force microscopy (AFM) is a type of scanning probe microscopy that can analyze topographic details of various specimens like ceramics, glass, polymers, and biological samples. AFM offers over 1000 times more resolution than the optical imaging system. Images generated from AFM are three-dimensional surface profiles, offering an advantage over the flat, two-dimensional images from other imaging techniques.
The AFM Probe
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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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Studying the Cytoskeleton01:17

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The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary...
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Overview of Microscopy Techniques01:22

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The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the...
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Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions
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High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic

Pascal D Odermatt, Arun Shivanandan, Hendrik Deschout

  • 1§National High Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee, Florida 32306 United States.

Nano Letters
|June 30, 2015
PubMed
Summary

This study introduces a novel correlated single molecule localization microscopy/atomic force microscopy (SMLM/AFM) technique. It enables precise localization of proteins within high-resolution AFM images, advancing nanoscale biological research.

Keywords:
Atomic force microscopy (AFM)correlative imagingdirect stochastic optical reconstruction microscopy (dSTORM)live cell imagingphotoactivated localization microscopy (PALM)single molecule localization microscopy (SMLM)

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Nanoscale characterization is crucial for understanding biological processes at the molecular level.
  • Atomic Force Microscopy (AFM) provides high-resolution topographical imaging of biological samples in aqueous environments.
  • Correlating nanoscale structure with specific protein function remains a significant challenge in modern biology.

Purpose of the Study:

  • To develop and validate a correlative microscopy technique combining Single Molecule Localization Microscopy (SMLM) with AFM.
  • To enable the localization of specific, labeled proteins within high-resolution AFM images of living samples.
  • To bridge the gap between structural information from AFM and functional insights from SMLM.

Main Methods:

  • Development of a dual-mode SMLM/AFM system.
  • Utilizing direct stochastic optical reconstruction microscopy (dSTORM)/AFM for cytoskeletal filament analysis.
  • Employing photoactivated light microscopy (PALM)/AFM for imaging bacterial and live mammalian cells.

Main Results:

  • Successful correlation of protein localization with 3D topography of F-actin filaments using dSTORM/AFM.
  • Acquisition of correlative images of bacterial cells in aqueous conditions using PALM/AFM.
  • Demonstration of the first correlative AFM/PALM imaging of live mammalian cells.

Conclusions:

  • The developed SMLM/AFM technique provides complementary nanoscale structural and functional information.
  • This correlative approach opens new avenues for investigating biological systems at the nanoscale.
  • The ability to localize specific proteins within AFM images enhances the understanding of molecular mechanisms in living cells.