Fluorescence Lifetime Correlation Spectroscopy (FLCS) is a variant of fluorescence correlation spectroscopy (FCS), which uses differences in fluorescence intensity decays to separate contributions of different fluorophore populations to FCS signal. Besides which, FLCS is a powerful tool to improve quality of FCS data by removing noise and distortion caused by scattered excitation light, detector thermal noise and detector afterpulsing. We are providing an overview of, to our knowledge, all published applications of FLCS. Although these are not numerous so far, they illustrate possibilities for the technique and the research topics in which FLCS has the potential to become widespread. Furthermore, we are addressing some questions which may be asked by a beginner user of FLCS. The last part of the text reviews other techniques closely related to FLCS. The generalization of the idea of FLCS paves the way for further promising application of the principle of statistical filtering of signals. Specifically, the idea of fluorescence spectral correlation spectroscopy is here outlined.

• MeSH
• Diffusion MeSH
• Fluorescent Dyes chemistry   MeSH
• Spectrometry, Fluorescence * MeSH
• Photons MeSH
• Liposomes chemistry   MeSH
• Models, Theoretical MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Fluorescent Dyes MeSH
• Liposomes MeSH

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

• MeSH
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Spectrometry, Fluorescence MeSH
• Fluorescence Recovery After Photobleaching MeSH
• HeLa Cells MeSH
• Kinetics MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• RNA Precursors metabolism   MeSH
• Ribonucleoproteins, Small Nuclear metabolism   physiology   MeSH
• RNA Splicing physiology   MeSH
• Spliceosomes metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Messenger MeSH
• RNA Precursors MeSH
• Ribonucleoproteins, Small Nuclear MeSH

Fluorescence correlation spectroscopy (FCS) is a single molecule technique used mainly for determination of mobility and local concentration of molecules. This review describes the specific problems of FCS in planar systems and reviews the state of the art experimental approaches such as 2-focus, Z-scan or scanning FCS, which overcome most of the artefacts and limitations of standard FCS. We focus on diffusion measurements of lipids and proteins in planar lipid membranes and review the contributions of FCS to elucidating membrane dynamics and the factors influencing it, such as membrane composition, ionic strength, presence of membrane proteins or frictional coupling with solid support.

• Keywords
• biomembranes, confocal microscopy, fluorescence fluctuation spectroscopy, giant unilamellar vesicles, lateral diffusion, supported lipid bilayers,
• MeSH
• Diffusion MeSH
• Spectrometry, Fluorescence * MeSH
• Microscopy, Confocal MeSH
• Lipid Bilayers chemistry   MeSH
• Membrane Lipids chemistry   MeSH
• Unilamellar Liposomes chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Lipid Bilayers MeSH
• Membrane Lipids MeSH
• Unilamellar Liposomes MeSH