Fluorescence cross-correlation spectroscopy


Fluorescence cross-correlation spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) was introduced by Eigen and Rigler in 1994 and experimentally realized by Schwille in 1997. It extends the fluorescence correlation spectroscopy (FCS) procedure by introducing high sensitivity for distinguishing fluorescent particles which have a similar diffusion coefficient. FCCS uses two species which are independently labelled with two spectrally separated fluorescent probes. These fluorescent probes are excited and detected by two different laser light sources and detectors commonly known as green and red respectively. Both laser light beams are focused into the sample and tuned so that they overlap to form a superimposed confocal observation volume.

The normalized cross-correlation function is defined for two fluorescent species G and R which are independent green, G and red, R channels as follows:

G_{GR}( au)=1+frac{}{}=frac{}{}

where differential fluorescent signals delta I_G at a specific time, t and delta I_R at a delay time, au later is correlated with each other.

Modeling

Cross-correlation curves are modeled according to a slightly more complicated mathematical function than applied in FCS. First of all, the effective superimposed observation volume in which the G and R channels form a single observation volume, V_{eff, RG} in the solution:

V_{eff, RG}=pi^{3/2}(omega_{xy,G}^2+omega_{xy,R}^2)(omega_{z,G}^2+omega_{z,R}^2)^{1/2}/2^{3/2}

where omega_{xy,G}^2 and omega_{xy,R}^2 are radial parameters and omega_{z,G} and omega_{z,R} are the axial parameters for the G and R channels respectively.

The diffusion time, au_{D,GR} for a doubly (G and R) fluorescent species is therefore described as follows:

au_{D,GR}=frac{omega_{xy,G}^2+omega_{xy,R}^2}{8D_{GR

where D_{GR} is the diffusion coefficient of the doubly fluorescent particle.

The cross-correlation curve generated from diffusing doubly labelled fluorescent particles can be modelled in separate channels as follows:

G_G( au)=1+frac{(Diff_k( au)+Diff_k( au))}{V_{eff, GR}(+)^2}

G_R( au)=1+frac{(Diff_k( au)+Diff_k( au))}{V_{eff, GR}(+)^2}

In the ideal case, the cross-correlation function is proportional to the concentration of the doubly labeled fluorescent complex:

G_GR( au)=1+frac{Diff_{GR}( au)}{V_{eff}(+)(+)}

with Diff_k( au)=frac{1}{(1+frac{ au}{ au_{D,i)(1+a^{-2}(frac{ au}{ au_{D,i)^{1/2

Contrary to FCS, the intercept of the cross-correlation curve does not yield information about the doubly labelled fluorescent particles in solution.

ee also

*Fluorescence correlation spectroscopy
*Dynamic light scattering
*Fluorescence spectroscopy
*Diffusion coefficient

External links

* [http://www.fcsxpert.com/classroom FCS Classroom]


Wikimedia Foundation. 2010.

Look at other dictionaries:

  • Fluorescence correlation spectroscopy — (FCS) is a common technique used by physicists, chemists, and biologists to experimentally characterize the dynamics of fluorescent species (e.g. single fluorescent dye molecules in nanostructured materials, autofluorescent proteins in living… …   Wikipedia

  • Spectroscopy — Analysis of white light by dispersing it with a prism is example of spectroscopy. Spectroscopy ( …   Wikipedia

  • spectroscopy — spectroscopist /spek tros keuh pist/, n. /spek tros keuh pee, spek treuh skoh pee/, n. the science that deals with the use of the spectroscope and with spectrum analysis. [1865 70; SPECTRO + SCOPY] * * * Branch of analysis devoted to identifying… …   Universalium

  • Nuclear magnetic resonance spectroscopy — A 900MHz NMR instrument with a 21.2 T magnet at HWB NMR, Birmingham, UK Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei… …   Wikipedia

  • Methods to investigate protein–protein interactions — There are many methods to investigate protein–protein interactions. Each of the approaches has its own strengths and weaknesses, especially with regard to the sensitivity and specificity of the method. A high sensitivity means that many of the… …   Wikipedia

  • List of materials analysis methods — List of materials analysis methods: Contents: Top · 0–9 · A B C D E F G H I J K L M N O P Q R S T U V W X Y Z μSR see Muon spin spectroscopy …   Wikipedia

  • Petra Schwille — (* 25. Januar 1968 in Sindelfingen) ist eine deutsche Biophysikerin. Inhaltsverzeichnis 1 Lebenslauf 2 Forschungsschwerpunkte 3 Ehrungen 4 …   Deutsch Wikipedia

  • Schwille — Petra Schwille (* 25. Januar 1968 in Sindelfingen) ist eine deutsche Physikerin. Inhaltsverzeichnis 1 Lebenslauf 2 Forschungsschwerpunkte 3 Wichtige Veröffentlichungen 4 Weblinks …   Deutsch Wikipedia

  • Molecular models of DNA — While this purified DNA precipitated in a water jug (left) appears to be a formless mass, nucleic acids actually possess intricate structure at the nanoscale (right). M …   Wikipedia

  • Lipid raft — organisation, region (1) is standard lipid bilayer, while region (2) is a lipid raft. The plasma membrane of cells is made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains termed lipid… …   Wikipedia


Share the article and excerpts

Direct link
Do a right-click on the link above
and select “Copy Link”

We are using cookies for the best presentation of our site. Continuing to use this site, you agree with this.