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Related Concept Videos

Overview of Microscopy Techniques01:22

Overview of Microscopy Techniques

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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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Super-resolution Fluorescence Microscopy01:37

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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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Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals
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Automated ExEm-spFRET Microscope.

Han Sun1,2, Chenshuang Zhang1,2, Ye Yuan1,2

  • 1Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou510631, China.

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
|February 21, 2022
PubMed
Summary
This summary is machine-generated.

We developed an automatic fluorescence resonance energy transfer (FRET) microscope for quantitative imaging in living cells. This robust system enables fast, one-click FRET measurements and reveals insights into protein interactions and calcium dynamics.

Keywords:
automatic FRET microscopeexcitation–emission spectraonline measurementuser-friendly interface

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Area of Science:

  • Biophysics
  • Cell Biology
  • Microscopy

Background:

  • Fluorescence resonance energy transfer (FRET) microscopy is crucial for studying molecular interactions in living cells.
  • Existing FRET methods can be complex and time-consuming to operate.
  • There is a need for automated, quantitative FRET imaging systems with high temporal resolution.

Purpose of the Study:

  • To develop an automated excitation-emission spectral unmixing-based FRET (ExEm-spFRET) microscope.
  • To achieve high time resolution for quantitative FRET imaging in living cells.
  • To validate the system's stability and apply it to investigate biological processes.

Main Methods:

  • Development of an automated ExEm-spFRET microscope with user-friendly software.
  • Integration of automatic background recognition, subtraction, and cell segmentation.
  • Quantitative FRET imaging in living cells expressing tandem constructs, FRET two-hybrid assays, and time-lapse imaging of Ca2+ influx.

Main Results:

  • The automated ExEm-spFRET microscope achieved a time resolution of 3.04 seconds.
  • Consistent and stable FRET imaging results were obtained for over 3 months.
  • The system revealed distinct Bcl-xL-Bad complex formations in healthy and apoptotic cells and monitored rapid Ca2+ influx.

Conclusions:

  • The developed automated ExEm-spFRET microscope provides a robust and quantitative platform for live-cell FRET imaging.
  • The system demonstrates high stability and accuracy for long-term studies.
  • This technology facilitates the investigation of dynamic molecular interactions and cellular processes, such as protein complex formation and calcium signaling.