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Enrico Gratton - One of the best experts on this subject based on the ideXlab platform.
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Laurdan fluorescence and phasor plots reveal the effects of a H2O2 bolus in NIH-3T3 fibroblast membranes dynamics and hydration.
Free radical biology & medicine, 2018Co-Authors: Leonel Malacrida, Enrico GrattonAbstract:Abstract Fluorescence spectroscopy, coupled with microscopy, opens new frontiers for the study of dynamic processes with high spatio-temporal resolution. The application of phasor plots to FLIM and hyperspectral imaging demonstrate unprecedented capabilities to study complex photophysics at the subcellular level. Using these approaches we studied the effects of an H2O2 bolus on NIH-3T3 membranes dynamics monitored by Laurdan fluorescence. Exposure of NIH-3T3 cells to a bolus of H2O2 modifies the cell membranes and, in particular, the plasma membrane in a complex manner. The Laurdan results reveal that the peroxide treatment decreases membrane fluidity but surprisingly increases dipolar relaxation around the excited probe. Using the Multidimensional-phasor approach we elucidated the complex photophysics of Laurdan incorporated into cell membrane after H2O2 exposure. The results indicate the occurrence of Laurdan fast-diffusion from gel↔ld phases in membranes exposed to a H2O2 bolus. An ad hoc hypothesis is presented to interpret the results in the context of H2O2 oxidative distress/eustress.
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A multidimensional phasor approach reveals Laurdan photophysics in NIH-3T3 cell membranes
Scientific Reports, 2017Co-Authors: Leonel Malacrida, David M Jameson, Enrico GrattonAbstract:Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. Among the dyes used in membrane studies, Laurdan has the advantage to be sensitive to the lipid composition as well as to membrane fluidity. The Laurdan spectrum is sensitive to the lipid composition and dipolar relaxation arising from water penetration, but disentangling lipid composition from membrane fluidity can be obtained if time resolved spectra could be measured at each cell location. Here we describe a method in which spectral and lifetime information obtained in different measurements at the same plane in a cell are used in the phasor plot providing a solution to analyze multiple lifetime or spectral data through a common visualization approach. We exploit a property of phasor plots based on the reciprocal role of the phasor plot and the image. In the phasor analysis each pixel of the image is associated with a phasor and each phasor maps to pixels and features in the image. In this paper the lifetime and spectral fluorescence data are used simultaneously to determine the contribution of polarity and dipolar relaxations of Laurdan in each pixel of an image.
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Laurdan identifies different lipid membranes in eukaryotic cells
2014Co-Authors: Enrico Gratton, Michelle A. DigmanAbstract:Laurdan Identifies Different Lipid Membranes in Eukaryotic Cells Enrico Gratton and Michelle A. Digman CONTENTS 13.1 Introduction 283 13.1.1 Spectroscopic Properties of Laurdan 283 13.2 The Phasor Approach to Spectral and Lifetime Analysis 287 13.3 The Lifetime Phasor Transformation and Its Interpretation 289 13.4 Results of the Analysis of the Emission of Laurdan Using Spectral and Lifetime Phasors in GUVs Model Systems 291 13.5 The Lifetime Phasor for Laurdan in GUVs 292 13.6 Live Cell Membrane Fluidity 295 13.6.1 Spectral Phasors 295 13.6.2 Lifetime Phasors in Live 3T3 Cells 297 13.7 Conclusions and Further Considerations 299 13.8 Methods 300 13.8.1 Preparation of the GUVs 300 13.8.2 NIH3T3 Cell Cultures 301 13.8.3 FLIM Analysis 301 13.8.4 Spectral Analysis 301 Acknowledgment 301 References 302 13.1 INTRODUCTION 13.1.1 S pectroscopic P roperties of L aurdan There are several commonly used approaches for the study of membrane properties of live cells based on fluorescence probes. In one approach, lipids with specific fluores- cent markers are incorporated in the cell membranes. The advantage of this approach is that it is possible to study the membrane distribution of specific lipids. However, when the aim of the study is to detect membrane microdomains independently of their
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Laurdan fluorescence lifetime discriminates cholesterol content from changes in fluidity in living cell membranes
Biophysical Journal, 2013Co-Authors: Ottavia Golfetto, Elizabeth Hinde, Enrico GrattonAbstract:Detection of the fluorescent properties of Laurdan has been proven to be an efficient tool to investigate membrane packing and ordered lipid phases in model membranes and living cells. Traditionally the spectral shift of Laurdan’s emission from blue in the ordered lipid phase of the membrane (more rigid) toward green in the disordered lipid phase (more fluid) is quantified by the generalized polarization function. Here, we investigate the fluorescence lifetime of Laurdan at two different emission wavelengths and find that when the dipolar relaxation of Laurdan’s emission is spectrally isolated, analysis of the fluorescence decay can distinguish changes in membrane fluidity from changes in cholesterol content. Using the phasor representation to analyze changes in Laurdan’s fluorescence lifetime we obtain two different phasor trajectories for changes in polarity versus changes in cholesterol content. This gives us the ability to resolve in vivo membranes with different properties such as water content and cholesterol content and thus perform a more comprehensive analysis of cell membrane heterogeneity. We demonstrate this analysis in NIH3T3 cells using Laurdan as a biosensor to monitor changes in the membrane water content during cell migration.
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Laurdan Spectral Phasor Detects Membrane Micro-Heterogeneity and Lipid Domains in Live Cells
Biophysical Journal, 2013Co-Authors: Ottavia Golfetto, Elizabeth Hinde, Enrico GrattonAbstract:The fluorescent probe Laurdan is sensitive to membrane lateral packing and lipid order. Laurdan is commonly used in model and biological membranes to report on different lipid phases. Here we describe a novel approach to detect Laurdan spectral changes due to membrane packing in vivo: the Laurdan spectral phasor. By Fourier transformation of Laurdan's spectrum into a spectral phasor we obtain two coordinates for each pixel of an image. The linear combination of two spectral components always falls in the line joining the phasors representing the two components, which allows quantification of the relative contribution of the different lipid phases. If, however, there is a different environment for Laurdan in a pixel, the phasor of that pixel cannot be represented by the linear combination of the phasor of two components. Therefore, non-linear environment interactions can be immediately detected. We use this approach to perform a comprehensive analysis of membrane heterogeneity in NIH3T3 live cells. We take advantage of a greater sensitivity, compared to conventional techniques, and we are able to detect highly packed micro-domains and to monitor changes in membrane packing due to acute and chronic cholesterol manipulation in vivo. Work supported in part by NIH-P41 P41-RRO3155, 8P41GM103540 and P50-GM076516.
Annarina Ambrosini - One of the best experts on this subject based on the ideXlab platform.
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Idiopathic infertility: susceptibility of spermatozoa to in‐vitro capacitation, in the presence and the absence of palmitylethanolamide (a homologue of anandamide), is strongly correlated with membrane polarity studied by Laurdan fluorescence
Molecular human reproduction, 2003Co-Authors: Annarina Ambrosini, Giovanna Zolese, Michal Wozniak, D. Genga, M. Boscaro, F. Mantero, G. BalerciaAbstract:Capacitation is a widely investigated process, which induces sperm plasma membrane changes resulting in its increased affinity for the zona pellucida. The fluorescent probe Laurdan, localized only within the plasma membrane of spermatozoa, is particularly useful to evaluate bilayer polarity in this part of the cell. According to a previous study, sperm membranes from oligozoospermic and some normozoospermic subjects (defined according to World Health Organization criteria), are characterized by low polarity (high Laurdan exGP(340)), while the spermatozoa from the remaining normozoospermic men show a larger polarity (low exGP(340)). In this paper, Laurdan was used to study membrane changes occurring during in-vitro capacitation, on sperm membranes from oligozoospermic and normozoospermic subjects. Results indicated that cells with high exGP(340) show a different susceptibility to Ca(2+)-induced capacitation in vitro, as compared with cells with low exGP(340). Palmitylethanolamide, physiologically present in human reproductive tracts, affects the time-course of in-vitro capacitation, increasing the rate of this process only in the cells with a lower membrane polarity.
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Laurdan fluorescence a simple method to evaluate sperm plasma membrane alterations
Fertility and Sterility, 2001Co-Authors: Annarina Ambrosini, Giovanna Zolese, Enrico Bertoli, Giancarlo Balercia, Giorgio Arnaldi, Franco ManteroAbstract:Abstract Objective: To determine, by a simple fluorescence method, sperm plasma membrane alterations related with changes of lipid bilayer that, together with routine semen analysis, could help to elucidate the causes of the unexplained male infertility problems. Design: Pilot study. Setting: Andrology laboratory and biochemistry institute, medical school. Patient(s): Men whose semen was studied for infertility problems. Intervention(s): No therapeutic intervention was performed on patients. Main Outcome Measure(s): Presence of spermatozoa plasma membrane alterations evidenced by evaluation of Laurdan fluorescence Generalized Polarization (GP) and reported as a function of increasing cell concentration, spermatozoa total motility, linear speed, and vitality. Result(s): Reporting GP values as a function of increasing sperm cell concentration, it is evident that the samples are distributed in two distinct areas: at >32 × 10 6 cells per milliliter, mean GP value was 0.303 ± 0.015, whereas for lower sperm cell concentrations, the mean GP was 0.365 ± 0.026 ( P 6 cells per milliliter) show high GP values. Conclusion(s): Laurdan fluorescence can be used as a simple method to evaluate spermatozoa plasma membrane alterations, particularly in a group of infertile men presenting normal semen parameters. In these samples, Laurdan could be used as a simple tool for infertility assessment. In fact, it is known that compositional and physicochemical alterations of bilayer features can be important for the fertilizing ability of spermatozoa because they are necessary for a proper physiological membrane activity.
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Laurdan∗ fluorescence: a simple method to evaluate sperm plasma membrane alterations ☆
Fertility and sterility, 2001Co-Authors: Annarina Ambrosini, Giovanna Zolese, Enrico Bertoli, Giancarlo Balercia, Giorgio Arnaldi, Franco ManteroAbstract:Abstract Objective: To determine, by a simple fluorescence method, sperm plasma membrane alterations related with changes of lipid bilayer that, together with routine semen analysis, could help to elucidate the causes of the unexplained male infertility problems. Design: Pilot study. Setting: Andrology laboratory and biochemistry institute, medical school. Patient(s): Men whose semen was studied for infertility problems. Intervention(s): No therapeutic intervention was performed on patients. Main Outcome Measure(s): Presence of spermatozoa plasma membrane alterations evidenced by evaluation of Laurdan fluorescence Generalized Polarization (GP) and reported as a function of increasing cell concentration, spermatozoa total motility, linear speed, and vitality. Result(s): Reporting GP values as a function of increasing sperm cell concentration, it is evident that the samples are distributed in two distinct areas: at >32 × 10 6 cells per milliliter, mean GP value was 0.303 ± 0.015, whereas for lower sperm cell concentrations, the mean GP was 0.365 ± 0.026 ( P 6 cells per milliliter) show high GP values. Conclusion(s): Laurdan fluorescence can be used as a simple method to evaluate spermatozoa plasma membrane alterations, particularly in a group of infertile men presenting normal semen parameters. In these samples, Laurdan could be used as a simple tool for infertility assessment. In fact, it is known that compositional and physicochemical alterations of bilayer features can be important for the fertilizing ability of spermatozoa because they are necessary for a proper physiological membrane activity.
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THE EFFECT OF N-ACYL ETHANOLAMINES ON PHOSPHATIDYLETHANOLAMINE PHASE TRANSITIONS STUDIED BY Laurdan GENERALISED POLARISATION
Chemistry and physics of lipids, 1994Co-Authors: Annarina Ambrosini, Michal Wozniak, Enrico Bertoli, Fabio Tanfani, Giovanna ZoleseAbstract:Abstract The effect of N-lauroylethanolamine (N-LEA) and N-oleoylethanolamine (N-OEA) on the thermal behaviour of fully hydrated egg phosphatidylethanolamine (TPE) was investigated by the steady-state fluorescence of 2-dimethylamino-(6-lauroyl)-naphtalene (Laurdan) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). The parameter generalised polarisation (GP), calculated by exciting Laurdan at 340 and 410 nm, revealed the gel to liquid crystalline lamellar (Lα) as well as the Lα to inverse hexagonal (HII) phase transitions of TPE. The Lα to HII phase transition was not detected in TPE/N-OEA system, probably because of the formation of an intermediate Q224 cubic phase. The formation of Q224 phase in TPE/N-OEA and TPE/N-LEA systems was previously demonstrated by X-ray diffraction, but neither Laurdan generalised polarisation nor TMA-DPH steady-state fluorescence anisotropy measurements revealed the presence of this phase. It is suggested that the lack of detection of the cubic phase is probably due to the similarity in dynamic characteristics and hydration levels of phospholipid headgroups in the bilayer and cubic phases.
Luis A. Bagatolli - One of the best experts on this subject based on the ideXlab platform.
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Measuring molecular order for lipid membrane phase studies: Linear relationship between Laurdan generalized polarization and deuterium NMR order parameter.
Biochimica et biophysica acta. Biomembranes, 2019Co-Authors: Sherry See Wai Leung, Luis A. Bagatolli, Jonathan R. Brewer, Jenifer ThewaltAbstract:Abstract Two dimensional phase separation in lipid membranes and cell membranes is of interest to biology because of the idea of membrane rafts — compositionally heterogeneous liquid crystal domains with cellular functions. Few quantitative tools exist for characterizing and differentiating coexisting phases on a molecular scale. Lipid acyl chain order can be measured directly using deuterium nuclear magnetic resonance spectroscopy (2H NMR), or inferred using fluorescence microscopy along with the environment-sensitive probe Laurdan. We found a linear relationship between the 2H NMR order parameter and Laurdan generalized polarization. This observed correlation supports the idea that lipid chain order is tightly associated with the amount and dynamics of water molecules at the glycerol backbone level of the membrane.
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Enzymatic studies on planar supported membranes using a widefield fluorescence Laurdan Generalized Polarization imaging approach.
Biochimica et biophysica acta. Biomembranes, 2017Co-Authors: Jonathan R. Brewer, Henrik Seir Thoke, Roberto P. Stock, Luis A. BagatolliAbstract:We introduce a custom-built instrument designed to perform fast Laurdan Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate Laurdan GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of Laurdan GP imaging to explore this type of membrane phenomena.
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Effects of Fluorescent Probes on Lipid Membrane Physical Properties
Biophysical Journal, 2014Co-Authors: Sherry S.w. Leung, Jenifer Thewalt, Jonathan R. Brewer, Luis A. BagatolliAbstract:It is currently accepted that nano-scale lateral compositional heterogeneity plays functional roles in cell membranes. Ordered and disordered lipid phases can coexist at appropriately chosen compositions, temperatures and pressures. Fluorescence is a popular family of techniques used to study membranes, however recent systematic studies show that fluorescent probe behaviour can be altered by membrane composition, probe concentration, and the presence of other probes. Using deuterium nuclear magnetic resonance spectroscopy (2H NMR), we found that trace amounts of the carbocyanine probe DiIC12 are enough to alter phase coexistence behaviour of 30:30:35 DPPC-D62:DOPC:cholesterol membranes, while other probes like Laurdan, Naphthopyrene, and another carbocyanine probe DiOC18, did not affect the membrane appreciably. Laurdan is particularly well suited to the study of phase separation in lipid membranes. It partitions equally well into ordered and disordered lipid phases, and displays a phase-dependent emission spectral shift. Laurdan general polarization (GP) parameter, which characterizes said emission spectral shift, has been used to characterize membrane fluidity. We examine the relationship between Laurdan GP and 2H NMR order parameters.
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Laurdan fluorescence properties in membranes: a journey from the fluorometer to the microscope
Springer Series on Fluorescence, 2012Co-Authors: Luis A. BagatolliAbstract:After 32 years since its introduction, the particular fluorescence properties of 6-lauroyl-2-(dimethylamino)-naphtalene (Laurdan) in model and biological membranes are revisited. This review includes a historical perspective about the design, synthesis and initial description of the probe’s fluorescent properties, a discussion about the proposed mechanism of Laurdan sensitivity to membrane lateral packing, and a detailed description of the definition of the Generalized Polarization function. This article includes as well examples of the different experimental strategies involving Laurdan in model and biological membranes, using both bulk fluorescence spectroscopy measurements and spatially resolved information from fluorescence microscopy. The value of this probe in the study of membrane structure and dynamics is reflected in more than 330 papers reported in the existing literature.
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Giant vesicles, Laurdan, and two-photon fluorescence microscopy: evidence of lipid lateral separation in bilayers.
Methods in enzymology, 2003Co-Authors: Luis A. Bagatolli, Susana A Sanchez, Theodore L. Hazlett, Enrico GrattonAbstract:Publisher Summary This chapter describes giant vesicles, Laurdan, and two-photon fluorescence microscopy. The combination of Laurdan, giant unilamellar vesicles (GUVs), and two-photon fluorescence microscopy has been extremely useful in producing a microscopic picture of lipid-phase coexistence in the GUV bilayer model system. Laurdan is a unique probe, giving simultaneous information about morphology and phase state of lipid domains from fluorescence images. The critical issues in membrane biophysics today are centered on the molecular dynamics of the bilayer structure. The interplay between lipids that results in the formation of domains on a bilayer surface and the interactions among these domains and relevant membrane-associated biomolecules is of particular interest. The advantages of using a microscope as the optical arrangement are clear. The light collection efficiency of a well-designed microscope is greatly enhanced over other optical arrangements. In addition, the flexibility of fluorescence microscopes creates for the spectroscopist a malleable optical compartment that can be designed and readily redesigned as needed.
M. Teresa Lamy - One of the best experts on this subject based on the ideXlab platform.
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A closer look into Laurdan as a probe to monitor cationic DODAB bilayers
Journal of Photochemistry and Photobiology A: Chemistry, 2019Co-Authors: Marcos K. Masukawa, Cíntia C. Vequi-suplicy, Evandro L. Duarte, M. Teresa LamyAbstract:Abstract Laurdan is a fluorescent molecule largely used to probe the lipid packing and hydration of membranes. In phospholipid bilayers that undergo the gel-fluid thermal transition, Laurdan fluorescent band displays a significant red shift upon the transition. However, Laurdan in DODAB bilayers does not show this characteristic shift of the emission band. Hence, the analysis of the membrane fluidity through the so-called Generalized Polarization (GP) cannot be used with DODAB. Nevertheless, both static and time resolved fluorescence attest that the Laurdan fluorescence in DODAB, like in phospholipids, is composed of two emission bands. The emission bands are associated with excited states with different lifetimes, and the relative intensity of the bands is sensitive to the bilayer thermal phase. Analyses were performed by decomposing the emission spectrum into two Gaussian bands and by computing the Decay Associated Spectra (DAS), the latter with time resolved fluorescence. We conclude that Laurdan in DODAB bilayers is in a shallower position as compared with the probe in phospholipids, possibly due to the small DODAB head group. Hence, Laurdan could be a useful probe to monitor interactions at the surface of DODAB bilayers, for instance with genetic material, quite often associated with cationic vesicles in lipoplexes, with broad medical applications.
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New Insights on the Fluorescent Emission Spectra of Prodan and Laurdan
Journal of fluorescence, 2015Co-Authors: Cíntia C. Vequi-suplicy, Kaline Coutinho, M. Teresa LamyAbstract:Prodan and Laurdan are fluorescent probes largely used in biological systems. They were synthetized to be sensitive to the environment polarity, and their fluorescent emission spectrum shifts around 120 nm, from cyclohexane to water. Although accepted that their emission spectrum is composed by two emission bands, the origin of these two bands is still a matter of discussion. Here we analyze the fluorescent spectra of Prodan and Laurdan in solvents of different polarities, both by decomposing the spectrum into two Gaussian bands and by computing the Decay Associated Spectra (DAS), the latter with time resolved fluorescence. Our data show that the intensity of the lower energy emission band of Prodan and Laurdan (attributed, in the literature, to the decay of a solvent relaxed state) is higher in cyclohexane than in water, showing a decrease as the polarity of the medium increases. Moreover, in all solvents studied here, the balance between the two emission bands is not dependent on the temperature, strongly suggesting two independent excited states. Both bands were found to display a red shift as the medium polarity increases. We propose here a new interpretation for the two emission bands of Prodan and Laurdan in homogeneous solvents: they would be related to the emission of two independent states, and not to a pair of non-relaxed and solvent relaxed states.
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Laurdan Spectrum Decomposition as a Tool for the Analysis of Surface Bilayer Structure and Polarity: a Study with DMPG, Peptides and Cholesterol
Journal of Fluorescence, 2010Co-Authors: Aline D. Lúcio, Cíntia C. Vequi-suplicy, Roberto M. Fernandez, M. Teresa LamyAbstract:The highly hydrophobic fluorophore Laurdan (6-dodecanoyl-2-(dimethylaminonaphthalene)) has been widely used as a fluorescent probe to monitor lipid membranes. Actually, it monitors the structure and polarity of the bilayer surface, where its fluorescent moiety is supposed to reside. The present paper discusses the high sensitivity of Laurdan fluorescence through the decomposition of its emission spectrum into two Gaussian bands, which correspond to emissions from two different excited states, one more solvent relaxed than the other. It will be shown that the analysis of the area fraction of each band is more sensitive to bilayer structural changes than the largely used parameter called Generalized Polarization, possibly because the latter does not completely separate the fluorescence emission from the two different excited states of Laurdan. Moreover, it will be shown that this decomposition should be done with the spectrum as a function of energy, and not wavelength. Due to the presence of the two emission bands in Laurdan spectrum, fluorescence anisotropy should be measured around 480 nm, to be able to monitor the fluorescence emission from one excited state only, the solvent relaxed state. Laurdan will be used to monitor the complex structure of the anionic phospholipid DMPG (dimyristoyl phosphatidylglycerol) at different ionic strengths, and the alterations caused on gel and fluid membranes due to the interaction of cationic peptides and cholesterol. Analyzing both the emission spectrum decomposition and anisotropy it was possible to distinguish between effects on the packing and on the hydration of the lipid membrane surface. It could be clearly detected that a more potent analog of the melanotropic hormone α-MSH (Ac-Ser^1-Tyr^2-Ser^3-Met^4-Glu^5-His^6-Phe^7-Arg^8-Trp^9-Gly^10-Lys^11-Pro^12-Val^13-NH_2) was more effective in rigidifying the bilayer surface of fluid membranes than the hormone, though the hormone significantly decreases the bilayer surface hydration.
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Laurdan in Fluid Bilayers: Position and Structural Sensitivity
Journal of fluorescence, 2006Co-Authors: Cíntia C. Vequi-suplicy, Carlos R. Benatti, M. Teresa LamyAbstract:Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41 degrees C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel-fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.
Franco Mantero - One of the best experts on this subject based on the ideXlab platform.
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Laurdan fluorescence a simple method to evaluate sperm plasma membrane alterations
Fertility and Sterility, 2001Co-Authors: Annarina Ambrosini, Giovanna Zolese, Enrico Bertoli, Giancarlo Balercia, Giorgio Arnaldi, Franco ManteroAbstract:Abstract Objective: To determine, by a simple fluorescence method, sperm plasma membrane alterations related with changes of lipid bilayer that, together with routine semen analysis, could help to elucidate the causes of the unexplained male infertility problems. Design: Pilot study. Setting: Andrology laboratory and biochemistry institute, medical school. Patient(s): Men whose semen was studied for infertility problems. Intervention(s): No therapeutic intervention was performed on patients. Main Outcome Measure(s): Presence of spermatozoa plasma membrane alterations evidenced by evaluation of Laurdan fluorescence Generalized Polarization (GP) and reported as a function of increasing cell concentration, spermatozoa total motility, linear speed, and vitality. Result(s): Reporting GP values as a function of increasing sperm cell concentration, it is evident that the samples are distributed in two distinct areas: at >32 × 10 6 cells per milliliter, mean GP value was 0.303 ± 0.015, whereas for lower sperm cell concentrations, the mean GP was 0.365 ± 0.026 ( P 6 cells per milliliter) show high GP values. Conclusion(s): Laurdan fluorescence can be used as a simple method to evaluate spermatozoa plasma membrane alterations, particularly in a group of infertile men presenting normal semen parameters. In these samples, Laurdan could be used as a simple tool for infertility assessment. In fact, it is known that compositional and physicochemical alterations of bilayer features can be important for the fertilizing ability of spermatozoa because they are necessary for a proper physiological membrane activity.
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Laurdan∗ fluorescence: a simple method to evaluate sperm plasma membrane alterations ☆
Fertility and sterility, 2001Co-Authors: Annarina Ambrosini, Giovanna Zolese, Enrico Bertoli, Giancarlo Balercia, Giorgio Arnaldi, Franco ManteroAbstract:Abstract Objective: To determine, by a simple fluorescence method, sperm plasma membrane alterations related with changes of lipid bilayer that, together with routine semen analysis, could help to elucidate the causes of the unexplained male infertility problems. Design: Pilot study. Setting: Andrology laboratory and biochemistry institute, medical school. Patient(s): Men whose semen was studied for infertility problems. Intervention(s): No therapeutic intervention was performed on patients. Main Outcome Measure(s): Presence of spermatozoa plasma membrane alterations evidenced by evaluation of Laurdan fluorescence Generalized Polarization (GP) and reported as a function of increasing cell concentration, spermatozoa total motility, linear speed, and vitality. Result(s): Reporting GP values as a function of increasing sperm cell concentration, it is evident that the samples are distributed in two distinct areas: at >32 × 10 6 cells per milliliter, mean GP value was 0.303 ± 0.015, whereas for lower sperm cell concentrations, the mean GP was 0.365 ± 0.026 ( P 6 cells per milliliter) show high GP values. Conclusion(s): Laurdan fluorescence can be used as a simple method to evaluate spermatozoa plasma membrane alterations, particularly in a group of infertile men presenting normal semen parameters. In these samples, Laurdan could be used as a simple tool for infertility assessment. In fact, it is known that compositional and physicochemical alterations of bilayer features can be important for the fertilizing ability of spermatozoa because they are necessary for a proper physiological membrane activity.