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Related Concept Videos

Atomic Fluorescence Spectroscopy01:29

Atomic Fluorescence Spectroscopy

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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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UV–Vis Spectroscopy of Conjugated Systems01:32

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Organic compounds with conjugated double bonds show strong absorption features in the UV–visible region of the electromagnetic spectrum attributed to π → π* electronic excitations. Generally, a UV–vis absorption spectrum is recorded as a plot of absorbance vs wavelength. The wavelength of maximum absorbance, which manifests as a peak in the absorption spectrum, is denoted as λmax.
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2D NMR: Homonuclear Correlation Spectroscopy (COSY)01:06

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Homonuclear correlation spectroscopy, or COSY, is a 2-dimensional NMR technique that provides information about coupled protons. Typically, the geminal and vicinal coupling are observed. For example, consider the COSY spectrum of ethyl acetate, where its 1D proton NMR spectrum is plotted along the vertical and horizontal axes with their corresponding chemical shift scale. Three spots on the diagonal corresponding to the three peaks in the 1D proton spectrum are called diagonal peaks. The COSY...
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Diffusion01:12

Diffusion

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Diffusion is the passive movement of substances down their concentration gradients—requiring no expenditure of cellular energy. Substances, such as molecules or ions, diffuse from an area of high concentration to an area of low concentration in the cytosol or across membranes. Eventually, the concentration will even out, with the substance moving randomly but causing no net change in concentration. Such a state is called dynamic equilibrium, which is essential for maintaining overall...
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Correlations02:20

Correlations

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Correlation means that there is a relationship between two or more variables (such as ice cream consumption and crime), but this relationship does not necessarily imply cause and effect. When two variables are correlated, it simply means that as one variable changes, so does the other. We can measure correlation by calculating a statistic known as a correlation coefficient. A correlation coefficient is a number from -1 to +1 that indicates the strength and direction of the relationship between...
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Correlation and Causation01:27

Correlation and Causation

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Statistical tests can calculate whether there is a relationship, or correlation, between independent and dependent variables. An indirect relationship of the variables signifies a correlation, while a direct relationship shows causation. If it is determined that no connection exists between the variables, then the correlation is a coincidence.
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Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching
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Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems.

Cameron Hodges1, Rudra P Kafle1,2, J Damon Hoff1

  • 1Department of Biophysics, University of Michigan, 930 N. University Ave., Ann Arbor, MI, 4809-1055, USA.

Journal of Fluorescence
|January 26, 2018
PubMed
Summary
This summary is machine-generated.

This study introduces a computational method to correct for photobleaching artifacts in Fluorescence Correlation Spectroscopy (FCS). This technique enables accurate measurements of molecular diffusion in challenging biological environments like living cells.

Keywords:
Cellular transportFluorescence correlation spectroscopyLive-cell microscopyPhotobleachingSub-diffusion

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Area of Science:

  • Biophysics
  • Cellular Biology
  • Spectroscopy

Background:

  • Fluorescence Correlation Spectroscopy (FCS) is vital for studying molecular diffusion and aggregation in cells.
  • Cellular environments present challenges like crowding and photobleaching, limiting FCS utility.
  • Artifacts from photobleaching hinder quantitative analysis of molecular dynamics.

Purpose of the Study:

  • To develop a computational method to correct for photobleaching in FCS data.
  • To enable accurate quantitative analysis of molecular diffusion in challenging cellular environments.
  • To overcome limitations of FCS in living cells due to photobleaching.

Main Methods:

  • A novel method to compute time correlation functions using weighted photon arrival times.
  • Computational compensation for photobleaching effects during data analysis.
  • Validation using numerical simulations and experimental data from model solutions.

Main Results:

  • Successfully removed photobleaching effects from correlation functions.
  • Recovered quantitatively accurate mean-square displacements of fluorophores.
  • Demonstrated improved accuracy, especially when accounting for excitation volume deviations.

Conclusions:

  • The developed method effectively corrects for photobleaching in FCS.
  • Enables quantitative FCS studies in demanding environments like living cells.
  • Advances the application of FCS for studying transport and aggregation processes in vivo.