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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

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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Overview of Electron Microscopy01:25

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The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
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Cryo-electron Microscopy01:28

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Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
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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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Electron Microscope Tomography and Single-particle Reconstruction01:07

Electron Microscope Tomography and Single-particle Reconstruction

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Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
Electron Tomography
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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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Related Experiment Video

Updated: May 24, 2025

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy
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Molecule Differentiation Encoding Microscopy to Dissect Dense Biomolecules in Cellular Nanoenvironments below Spatial

Siyue Fan1, Xinyin Li1, Huan Liu1

  • 1Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xianning West Road, Xi'an, Shaanxi, 710049, China.

Angewandte Chemie (International Ed. in English)
|March 4, 2025
PubMed
Summary

This study introduces a new microscopy method using DNA barcodes to count dense biomolecules within cells. This technique overcomes resolution limits, revealing nanoscale organization crucial for biological functions.

Keywords:
Cell imagingDNA labelingDigital quantitationMolecular crowdingMolecule differentiation

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Area of Science:

  • Cellular and Molecular Biology
  • Nanotechnology
  • Biophysics

Background:

  • Cellular biomolecules are densely organized at the nanoscale, controlling essential biological processes.
  • Current microscopy techniques struggle to resolve the precise spatial distribution and copy numbers of these dense biomolecules.

Purpose of the Study:

  • To develop a novel method for digitally quantifying dense biomolecules within cellular nanoenvironments.
  • To overcome the spatial resolution limitations of conventional microscopy for nanoscale biological analysis.

Main Methods:

  • Developed a molecule differentiation encoding microscopy (MDEM) technique using orthogonal tandem repeat DNA identifiers.
  • Employed stochastic multiplexed reactions to barcode individual biomolecule copies with unique DNA sequences.
  • Created an algorithm for automated quantification of overlapping and individual molecular spots.

Main Results:

  • Successfully visualized and quantified dense distributions of RNAs, DNA epigenetic modifications, and cell surface glycans/glycoRNAs.
  • Demonstrated that various biomolecules exhibit dense organization within crowded cellular nanoenvironments.
  • Quantified an average of 17% of U1 glycoRNA copies localized to cell surface nanoenvironments.

Conclusions:

  • The developed MDEM strategy enables digital quantitative visualization of biomolecules below microscopy's spatial resolution.
  • This method provides critical insights into the functional implications of dense biomolecular organization in cellular nanoenvironments.