fluorescence spectroscopy
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Springer series on fluorescence. Methods and applications, ISSN ISSN 1617-1306 1
xxv, 490 s. : il. ; 25 cm
- Klíčová slova
- Biochemie, Laboratorní technika,
- MeSH
- biochemie MeSH
- fluorescence MeSH
- fluorescenční mikroskopie metody MeSH
- fluorescenční spektrometrie MeSH
- klinické laboratorní techniky MeSH
- molekulární struktura MeSH
- proteiny chemie MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biochemie
2nd ed. xxiii, 698 s. : il.
- Klíčová slova
- Biochemie, Laboratorní technika,
- MeSH
- biochemie MeSH
- fluorescence MeSH
- fluorescenční protilátková technika MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- fyzika, biofyzika
xvii, 294 s. : il.
- Klíčová slova
- Laboratorní technika,
- MeSH
- fluorescence MeSH
- fluorescenční protilátková technika MeSH
- fluorescenční spektrometrie MeSH
- molekulární biologie metody MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biochemie
- fyzika, biofyzika
Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.
Chloroplast thylakoid membranes contain virtually all components of the energy-converting photosynthetic machinery. Their energized state, driving ATP synthesis, is enabled by the bilayer organization of the membrane. However, their most abundant lipid species is a non-bilayer-forming lipid, monogalactosyl-diacylglycerol; the role of lipid polymorphism in these membranes is poorly understood. Earlier 31P-NMR experiments revealed the coexistence of a bilayer and a non-bilayer, isotropic lipid phase in spinach thylakoids. Packing of lipid molecules, tested by fluorescence spectroscopy of the lipophilic dye, merocyanine-540 (MC540), also displayed heterogeneity. Now, our 31P-NMR experiments on spinach thylakoids uncover the presence of a bilayer and three non-bilayer lipid phases; time-resolved fluorescence spectroscopy of MC540 also reveals the presence of multiple lipidic environments. It is also shown by 31P-NMR that: (i) some lipid phases are sensitive to the osmolarity and ionic strength of the medium, (ii) a lipid phase can be modulated by catalytic hydrogenation of fatty acids and (iii) a marked increase of one of the non-bilayer phases upon lowering the pH of the medium is observed. These data provide additional experimental evidence for the polymorphism of lipid phases in thylakoids and suggest that non-bilayer phases play an active role in the structural dynamics of thylakoid membranes.
Studies of lateral diffusion are used for the characterization of the dynamics of biological membranes. One of the techniques that can be used for this purpose is fluorescence correlation spectroscopy (FCS), which belongs to the single-molecule techniques. Unfortunately, FCS measurements, when performed in planar lipid systems, are associated with a few sources of inaccuracy in the determination of the lateral diffusion coefficient. The main problems are related to the imperfect positioning of the laser focus relative to the plane of the sample. Another source of inaccuracy is the requirement for external calibration of the detection volume size. This protocol introduces a calibration-free method called Z-scan fluorescence correlation spectroscopy (Z-scan FCS), which is based on the determination of the diffusion time and particle number in steps along the optical (z-) axis by sequential FCS measurements. Z-scan FCS could be employed for diffusion measurements in planar membrane model systems-supported phospholipid bilayers (SPBs) and giant unilamellar vesicles (GUVs) and also in biological membranes. A result from measurements in SPBs is also presented in the protocol as a principle example of the Z-scan technique.
- MeSH
- buněčná membrána chemie metabolismus MeSH
- difuze MeSH
- fluidita membrány MeSH
- fluorescence MeSH
- fluorescenční spektrometrie přístrojové vybavení metody MeSH
- kalibrace MeSH
- lipidové dvojvrstvy analýza chemie MeSH
- membránové lipidy chemie MeSH
- teoretické modely MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Basic principles of fluorescence correlation spectroscopy (FCS) are explained. The method affords diffusion coefficients and concentrations of fluorescent-labeled species by studying temporary fluctuations of fluorescence signal caused by diffusion through the focal volume of confocal microscope. In dual-color FCS, cross-correlation of two signals from molecules labeled with two spectrally shifted dyes allows to observe interactions between the two molecules. It is also possible to distinguish between the signals of two dyes with different lifetimes. FCS uses pulsed laser excitation.
1 online zdroj
- MeSH
- fluorescence * MeSH
- fluorescenční spektrometrie * MeSH
- Publikační typ
- periodika MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- fyzika, biofyzika
- chemie, klinická chemie
... Heberle -- Kinetic Multichannel Spectroscopy of Biological Molecules. ... ... -- Resonance Energy Transfer on Fluorescence and Phosphorescence -- J. ... ... Carmona -- Resonance CARS Spectra of Fluorescent Protein dsRed 38 -- S. G. Kruglik, V. ... ... Fluorescence Study 111 -- Z. H. ZaWADA. a. MlCHNIK and Z. ... ... Study by Raman Spectroscopy. N. Failloux. /. bonnetj and M. ...
2 sv. (150 s., s. 151-274) : il., tab. ; 25 cm
