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Atomic Fluorescence Spectroscopy01:29

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Atomic fluorescence spectroscopy (AFS) is an analytical technique that involves the electronic transitions of atoms in a flame, furnace, or plasma being excited by electromagnetic (EM) radiation. When these atoms absorb energy, they become excited and subsequently release energy as they return to their original state. This emitted light, or "fluorescence," is observed at a right angle to the incident beam. Both absorption and emission processes transpire at distinct wavelengths, which...
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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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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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Fluorescence and phosphorescence are essential phenomena in fields like analytical chemistry, biological imaging, and materials science, where they detect molecular properties and visualize cellular structures. Understanding the variables that influence these luminescent behaviors is crucial for maximizing accuracy and efficiency in their applications. These variables can broadly be grouped into chemical structure, solvent properties, and external conditions, each playing a distinct role in...
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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Fluorescence correlation spectroscopy.

Mark A Hink1

  • 1Department Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy (LCAM), University of Amsterdam, Sciencepark 904, 1098 XH, Amsterdam, The Netherlands, m.a.hink@uva.nl.

Methods in Molecular Biology (Clifton, N.J.)
|November 14, 2014
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Summary
This summary is machine-generated.

Fluorescence fluctuation spectroscopy quantifies nanomolar molecules. This chapter details a protocol for measuring molecular interactions of signaling proteins within living cells using fluorescence cross-correlation spectroscopy.

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Area of Science:

  • Biophysics
  • Cellular Biology
  • Molecular Interactions

Background:

  • Fluorescence fluctuation spectroscopy (FFS) enables precise quantification of fluorescent molecules at nanomolar concentrations.
  • Understanding molecular interactions within living cells is crucial for deciphering cellular signaling pathways.

Purpose of the Study:

  • To introduce fluorescence fluctuation spectroscopy techniques.
  • To provide a detailed protocol for measuring molecular interactions of signaling proteins inside living cells.

Main Methods:

  • Utilizing fluorescence cross-correlation spectroscopy (FCS), a key FFS technique.
  • Implementing a comprehensive protocol with necessary background information for accurate measurements.

Main Results:

  • The chapter presents a method for quantifying molecular interactions at the nanomolar level within a cellular environment.
  • The protocol facilitates the measurement of specific protein-protein interactions in real-time.

Conclusions:

  • Fluorescence fluctuation spectroscopy, particularly FCS, is a powerful tool for studying molecular interactions in vivo.
  • The provided protocol enables researchers to investigate dynamic cellular processes involving signaling proteins.