A New Fluorescent Squaraine Probe for the Measurement of Membrane Polarity

The present study was undertaken to evaluate the sensitivity of newly synthesized squaraine dye 1 to the changes in lipid bilayer physical properties and compared it with the well-known dye 2. Partitioning of the dye 1 into lipid bilayer was found to be followed by significant increase of its fluorescence intensity and red-shift of emission maximum, while intensity of the dye 2 fluorescence increased only slightly on going from aqueous to lipidic environment. This suggests that dye 1 is more sensitive to the changes in membrane properties as compared to dye 2. Partition coefficients of the dye 1 have been determined for the model membranes composed of zwitterionic phospholipid phosphatidylcholine (PC) and its mixtures with positively charged detergent cetyltrimethylammonium bromide (CTAB), anionic phospholipid cardiolipin (CL), and sterol (Chol). The spectral responses of the dye 1 in different liposome media proved to correlate with the increase of bilayer polarity induced by Chol and CL or its decrease caused by CTAB. It was concluded that dye 1 can be used as fluorescent probe for examining membrane-related processes.     

Interctions of laurdan with membranes at high pressures

Abstract

The fluorescence emission spectra of Laurdan in egg yolk phosphatidylcholine vesicles were measured as a function of pressure. The results suggest that the long hydrocarbon side chain stabilizes Laurdan in bilayers at high pressures.     

Fluorescence labeling and interaction of atherogenic lipoproteins with cultured cells

We prepared lipoprotein (a) and LDL covalently labeled with either BODIPY or rhodamine. A dual wavelength method was used for the microscopic observation of both lipoproteins during their interaction with HepG2 cells. Since a large proportion of Lp(a) colocalized with LDL on the cell surface and inside the cells, it was concluded that Lp(a) uptake into cells is mediated by LDL via internalization of LDL.     

Review of Fluorescence Suppression Techniques in Raman Spectroscopy

Abstract

Raman spectroscopy is an important and powerful technique for analyzing the chemical composition of biological or nonbiological samples in many fields. A serious challenge frequently encountered in Raman measurements arises from the existence of the concurrent fluorescence background. The fluorescence intensity is normally several orders of magnitude larger than the Raman scattering signal, especially in biological samples. Such fluorescence background must be suppressed in order to obtain accurate Raman spectra. Several different techniques have been explored for this purpose. These techniques could be generally grouped into time-domain, frequency-domain, wavelength-domain, and computational methods in addition to various Raman enhancement techniques and other unconventional methods. This review briefly describes the fundamental principles of each group of methods, reports the most recent advances, and makes comparison across those major categories of techniques in terms of cost and performance in a hope to guide interested readers to select proper methods for specific applications.     

利用荧光法检测磷酸酯液压油的泄漏

提出了一种检测飞机液压油泄漏的方法--荧光法,以美孚磷酸酯液压油为例,实验验证了荧光法检测磷酸酯液压油泄漏的可行性。采用LS-55型荧光分光光度计以及实验室搭建系统对HyJet Ⅴ磷酸酯液压油以及Jet Oil Ⅱ、2197润滑油的荧光特性进行了分析。研究结果表明,可以用不同荧光发射光谱峰值来区分三种油,荧光法检测磷酸酯液压油泄漏是可行的。该技术可实现对飞机液压油泄漏的实时、在线、现场测量。     

Long-lived fluorescence probes for studying lipid dynamics: A review

A great many studies have focused on the heterogeneous packing of lipids in the bilayer matrix. However, less attention has been directed toward the temporal aspects of these lipid-lipid interactions. Studies of lipid packing fluctuations, or gel-fluid exchange, using fluorescence probe methodologies have been limited. This limitation arises from thesubmicrosecond time scale over which the fluctuations are expected to occur. Traditionally, dynamic studies of lipid bilayers have been restricted to the nanosecond time regime, and the submicrosecond time window has not been explored in any great depth by fluorescence methods, although persistent lipid dynamics has been evident. Probes with long fluorescence lifetimes (several hundred nanoseconds) have the potential to expand this important time window, providing information on gel-fluid exchange rates and insights into how important biological effectors such as proteins, cholesterol, and anesthetics affect or modulate these fluctuations. Using the long-lived fluorescence probe coronene, combined with time-resolved fluorescence methods geared toward microheterogeneity, we present a view of bilayer dynamics in an alternate time domain. Fluorescence probes are expected to inhabit an equilibrium between fluid and gel environments. Some probes remain in their respective environments throughout their excited-state lifetime, while others reside in surroundings that will change (i.e., melt). Long-lived fluorescence membrane probes can provide direct estimates of submicrosecond lipid fluctuation or melt rates. Simple Landau modeling leads to adistribution of melt rates and provides an attractive alternative to a simplercompartmental model where a unique lipid fluctuation of gel-fluid exchange rate is measured. Thedistribution model is probe independent (defined by thermodynamic quantities) and can be applied generally to the rotational motions of fluorescence probes embedded in the lipid bilayer.Abbreviations DMPC l--dimyristoylphosphatidylcholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DPPC l--dipalmitoylphospha-tidylcholine - DSC differential scanning calorimetry - EA fluorescence emission anisotropy - LUV large unilamellar vesicles - SUV small unilamellar vesicles - T_c lipid phase transition temperature     

Fluorescence anisotropy and light-scattering studies of the interaction of insulin with liposomes

The interaction between zinc-stabilized insulin and lecithin liposomal membranes was studied using DPH fluorescence anisotropy and light-scattering techniques. To ascertain a possible influence of a charge on the insulin molecule, experiments were performed at pH 4.5 (insulin possesses a positive charge) and at pH 7.4 (the charge of insulin is negative). Measurements at pH 4.5 revealed significant changes in scattered light intensity induced by the addition of insulin to lecithin liposomes. With increasing time of storage of liposomes the insulin effect became faster and more pronounced. At pH 7.4, significant changes in scattered light were registered only in the case of liposomes stored for 5 days. In these liposomes a peroxidation process of lecithin was revealed. No significant changes induced by insulin were observed in DPH fluorescence anisotropy either at pH 4.5 or at pH 7.4, which suggested the absence of an interaction of insulin with the hycrophobic core of liposomes. Thus, the observed changes in scattered light could be interpreted in terms of the insulin association to the liposomal surface in the case of phospholipid peroxidation and/or acidic pH.     

Video fluorescence microscopic techniques to monitor local lipid and phospholipid molecular order and organization in cell membranes during hypoxic injury

Digitized video microscopy is rapidly finding uses in a number of fields of biological investigation because it allows quantitative assessment of physiological functions in intact cells under a variety of conditions. In this review paper, we focus on the rationale for the development and use of quantitative digitized video fluorescence microscopic techniques to monitor the molecular order and organization of lipids and phospholipids in the plasma membrane of single living cells. These include (1) fluorescence polarization imaging microscopy, used to measure plasma membrane lipid order, (2) fluorescence resonance energy transfer (FRET) imaging microscopy, used to detect and monitor phospholipid domain formation, and (3) fluorescence quenching imaging microscopy, used to spatially map fluid and rigid lipid domains. We review both the theoretical as well as practical use of these different techniques and their limits and potential for future developments, and provide as an illustrative example their application in studies of plasma membrane lipid order and topography during hypoxic injury in rat hepatocytes. Each of these methods provides complementary information; in the case of hypoxic injury, they all indicated that hypoxic injury leads to a spatially and temporally heterogeneous alteration in lipid order, topography, and fluidity of the plasma membrane. Hypoxic injury induces the formation of both fluid and rigid lipid domains; the formation of these domains is responsible for loss of the plasma membrane permeability barrier and the onset of irreversible injury (cell death). By defining the mechanisms which lead to alterations in lipid and phospholipid order and organization in the plasma membrane of hypoxic cells, potential sites of intervention to delay, prevent, or rescue cells from hypoxic injury have been identified. Finally, we briefly discuss fluorescence lifetime imaging microscopy (FLIM) and its potential application for studies monitoring local lipid and phospholipid molecular order and organization in cell membranes.     

Fluorescence techniques for probing water penetration into lipid bilayers

Fluorescence spectroscopy can be used as a highly sensitive and localized probe for hydration in lipid bilayers. Water associates with the head-group region, where it participates in an interlipid network of hydrogen bonds. Deeper in the bilayer, water is contained within acyl-chain packing defects. Fluorescence methodology is available to probe both the interstitial and head-group hydration in lipid bilayers, and results are in good agreement with other techniques. Using fluorescence spectroscopic approaches, cholesterol is shown to dehydrate the acyl-chain region, while hydrating the head-group region. Membrane proteins appear to increase acyl-chain hydration at the protein-lipid interface. Overall fluorescence spectroscopic techniques may be most effective in studying the water content of lipid bilayers and especially of biological membranes.     

Inter- and Intramolecular Dynamics of Pyrenyl Lipids in Bilayer Membranes from Time-Resolved Fluorescence Spectroscopy

The analysis of time-resolved fluorescence of monopyrenyl phospholipid monomer and excimer emission is reviewed using a model based on the diffusion equation for bimolecular reactions in two-dimensional planar membrane bilayers. This aspect is illustrated by the analysis of a real experiment. The method can also be extended to short-range diffusion, which can be measured for dipyrenyl phospholipids in membrane bilayers. The latter aspect is illustrated by the analysis of a simulated experiment to show the experimental requirements to recover the desired parameters.