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Related Concept Videos

Confocal Fluorescence Microscopy01:16

Confocal Fluorescence Microscopy

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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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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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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins
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Microscope alignment using real-time Imaging FCS.

Daniel Y K Aik1, Thorsten Wohland2

  • 1Center for BioImaging Sciences, National University of Singapore, Singapore; Department of Chemistry, National University of Singapore, Singapore.

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|June 8, 2022
PubMed
Summary
This summary is machine-generated.

A new tool enables real-time data access for electron-multiplying charge-coupled device (EMCCD) and scientific complementary metal-oxide semiconductor (sCMOS) cameras. This accelerates fluorescence microscopy experiments and simplifies alignment for total internal reflection fluorescence microscopy (TIRFM) and single-plane illumination microscopy (SPIM).

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Scientific Instrumentation

Background:

  • Modern scientific cameras (EMCCD, sCMOS) offer high sensitivity and speed for fluorescence imaging.
  • Real-time data evaluation is crucial for maximizing the potential of these advanced imaging systems.
  • Current methods can be slow and complex, hindering rapid experimental progress.

Purpose of the Study:

  • To develop a direct camera-read-out tool for real-time fluorescence data access.
  • To simplify and accelerate alignment procedures in TIRFM and SPIM.
  • To facilitate faster and more accurate fluorescence-based experiments.

Main Methods:

  • Implementation of a direct camera-read-out software tool.
  • Integration with widely used EMCCD and sCMOS camera models.
  • Utilizing imaging fluorescence correlation spectroscopy for data evaluation.

Main Results:

  • The tool provides real-time access to camera data during recording.
  • Simplified and accurate alignment for TIRFM and SPIM setups.
  • Demonstrated acceleration of fluorescence experimental workflows.

Conclusions:

  • The developed tool enhances the usability of high-speed scientific cameras.
  • It serves as a foundation for automated data acquisition in advanced microscopy.
  • The tool is extensible to other camera models and imaging techniques.