Fluorescence and ESR studies on membrane oxidative damage by gamma radiation

Oxidative damage to cellular membranes critically controls the manifestation of cellular response to ionizing radiation. To gain further insight into the damaging mechanisms, we have investigated the effects of γ-radiation-generated free-radical-mediated peroxidative damage in egg yolk lecithin unilamellar liposomal membranes by employing 1,6-diphenyl-1,3,5-hexatriene (DPH). Alterations in lipid bilayer fluidity and malondialdehyde (MDA) formation were measured in irradiated liposomal membranes as a function of radiation dose (0.1-1 kGy). A relationship seems to exist between the degree of radiation-induced peroxidative damage and the magnitude of DPH fluorescence decay in irradiated membranes. Radiation-induced membrane rigidization and MDA formation were significantly reduced when α-tocopherol, a natural membrane antioxidant, was present in the liposomes suggesting an involvement of lipid free radicals in the mechanism of the damage process. The results of the present study have been compared with those obtained by the electron spin resonance (ESR) technique on human erythrocyte ghost membranes with spin-labeled phospholipids having the unique capability to sensitively report on the dynamic state of the lipid environment inside the bilayer membrane. Iodoacetamide and N-ethylmaleimide spin labels were used to investigate alterations in membrane proteins. These results have contributed to our understanding of mechanisms involved in radiation membrane oxidative damage in terms of lipid peroxidation, fluidity changes and involvement of -SH groups of membrane proteins. Combined use of fluorescence and ESR spin-label techniques is of potential interest in probing the deeper molecular mechanisms of radiation injury in cellular membranes for developing strategies to modify the radiation damage to cells.     

荧光偏振法研究脉冲电场对酿酒酵母细胞膜流动性影响

以DPH(1,6-二苯基-1,3,5-己三烯)为荧光探剂,采用荧光偏振法探讨了脉冲电场(0～25 kV·cm-1,0～266 ms)对酿酒酵母细胞膜流动性影响。经5 kV·cm-1电场处理后,酿酒酵母细胞膜的流动性显著减小,并且随电场强度和处理时间的增加而减小;通过平板计数法和紫外分光光度计法分别检测了脉冲电场对酿酒酵母细胞存活对数及膜通透性影响。结果显示,5 kV·cm-1虽然只能使少量的酵母致死,却能使酵母细胞膜的通透性显著增加,膜流动性显著降低。并且细胞的存活率随电场强度增大而减小,细胞膜的通透性随电场强度增大而增大。这表明细胞膜的流动性降低与细胞膜的通透性升高成正相关,与细胞的存活率成负相关。由此推测脉冲电场在对酿酒酵母灭菌过程中,细胞膜是其作用的一个关键位点,膜流动性减小,细胞膜通透性增强,是细胞死亡的主要原因。     

Study of plasma membrane heterogeneity using a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene [2-(3-(diphenylhexatriene)propanoyl)-3-palmitoyl-l-α-phosphatidylcholine]

The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH derivatives have been used to characterize structural and physicochemical properties of specific membrane domains. Steady-state and fluorescence decay measurements of three probes, DPH (1,6-diphenyl-1,3,5-hexatriene), TMA-DPH [1-(4-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene], and a phosphatidylcholine derivative of DPH, DPH-pPC [2-(3-(diphenylhexatriene)propanoyl)-3-pamitoyl-L--phosphatidyl choline], have been performed in erythrocyte membranes and in lymphocyte plasma membranes. The steady-state fluorescence polarization of the three probes showed a similar trend in both membranes. In fact either in erythrocyte or in lymphocyte plasma membranes the fluorescence polarization values of DPH-pPC and TMA-DPH were similar, but significantly higher with respect to DPH. A better characterization of erythrocyte and lymphocyte plasma membranes was possible by using fluorescence decay measurements. The data suggest the possible use of different DPH derivatives to characterize specific domains in biological membranes.     

Enhancement of ultrasonically induced cell damage by phthalocyanines in vitro

In this work, erythrocytes from carp were used as a nucleated cell model to test the hypothesis that the phthalocyanines (zinc - ZnPc and chloroaluminium -AlClPc) enhance ultrasonically induced damage in vitro. In order to confirm and complete our earlier investigation, the influence of ultrasound (US) and phthalocyanines (Pcs) on unresearched cellular components, was studied. Red blood cells were exposed to 1 MHz continuous ultrasound wave (0.61 and/or 2.44 W/cm²) in the presence or absence of phthalocyanines (3 μM). To identify target cell damage, we studied hemolysis, membrane fluidity and morphology of erythrocytes. To demonstrate the changes in the fluidity of plasma membrane we used the spectrofluorimetric methods using two fluorescence probes: 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5,-hexatriene (TMA-DPH) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The effect of US and Pcs on nucleated erythrocytes morphology was estimated on the basis of microscopic observation.The enhancement of ultrasonically induced membrane damage by both phthalocyanines was observed in case of hemolysis, and membrane surface fluidity, in comparison to ultrasound. The authors also observed changes in the morphology of erythrocytes. The obtained results support the hypothesis that the Pcs enhance ultrasonically induced cell damage in vitro.Furthermore, the influence of ultrasound on phthalocyanines (Pcs) in medium and in cells was tested. The authors observed changes in the phthalocyanines absorption spectra in the medium and the increase in the intensity of phthalocyanines fluorescence in the cells. These data can suggest changes in the structure of phthalocyanines after ultrasound action.     

荧光偏振法测定红细胞膜粘度在药学方面的应用

本文介绍荧光偏振法应用于“血瘀”的动物模型,观察用人参后血液红细胞膜粘度的变化,借以研究人参对于“血瘀”用药的药效作用.     

Hemolysis of Erythrocytes and Erythrocyte Membrane Fluidity Changes by New Lysosomotropic Compounds

This work contains the results of studies on the influence of newly synthesized lysosomotropic substances (lysosomotropes) on human erythrocytes. Six homologous series of the compounds differing in the alkyl chain length and counterions were studied. They were found to hemolyse erythrocytes and to change their osmotic resistance. The observed hemolytic effects were dependent both on the compounds structure (polar head dimension and alkyl chain length of compound) and its form (the kind of the counterion). In parallel, the influence of lysosomotropes on fluidity of the erythrocyte membrane was studied. Three different fluorescent probes were used; 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, p-toluenesulfonate (TMA-DPH) and 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan). Their anisotropy (DPH and TMA-DPH) or general polarization (laurdan) values after incorporation into ghost erythrocyte membranes were measured. The results obtained show that fluidity changes accompanied the effects observed in hemolytic experiments both quantitatively and qualitatively.     

Fluorescence evidence for cholesterol regular distribution in phosphatidylcholine and in sphingomyelin lipid bilayers

Our previous studies indicated that sterols (including cholesterol and dehydroergosterol) can be regularly distributed into hexagonal superlattices in the plane of liquid-crystalline phosphatidylcholine bilayers. It was suggested that regular and irregular regions coexist in the membrane. In the present study, we report supporting evidence for our sterol regular distribution model. We have examined the fractional concentration dependencies of dehydroergosterol (a naturally occurring cholesterol analogue) fluorescence intensity and lifetime in various phosphatidylcholine and sphingomyelin bilayers. Fluorescence intensity and lifetime dips have been observed at specific sterol mole fractions. At those mole fractions, the acrylamide quenching rate constant of dehydroergosterol fluorescence reaches a local maximum. Those mole fractions match the critical sterol mole fractions at which sterol molecules are expected to be regularly distributed into hexagonal superlattices. The results support the idea that the sterols in the regular region are embedded in the bilayer less deep than those in the irregular regions. We have also examined the fractional cholesterol concentration dependencies of diphenylhexatriene (DPH) fluorescence intensity, lifetime, and polarization in DMPC vesicles. DPH fluorescence intensity and polarization also exhibit distinct dips and peaks, respectively, at critical sterol mole fractions for hexagonal superlattices. However, DPH lifetime changes little with sterol mole fraction. As a comparison, the fluorescence properties of DHE and DPH behave differently in response to the formation of sterol regular distribution. Furthermore, finding evidence for sterol regular distribution in both phosphatidylcholine and sphingomyelin membranes raises the possibility that sterol regular distribution may occur within phospholipid/cholesterol enriched domains of real biological membranes.     

Time-gated microscopic energy transfer measurements for probing mitochondrial metabolism

Spectroscopic and microscopic methods for probing mitochondrial malfunction were established using cultivated endothelial cells from the calf aorta and various inhibitors of the respiratory chain, which is located at the inner mitochondrial membrane. Time-gated fluorescence spectroscopy was used to measure autofluorescence of the coenzyme NADH as well as “energy transfer efficacy” from excited NADH molecules (energy donor) to the mitochondrial marker rhodamine-123 (energy acceptor). Autofluorescence usually exhibited a weak increase after specific inhibition of enzyme complexes of the respiratory chain. In contrast, a pronounced increase in energy transfer efficacy was observed after inhibition of the same enzyme complexes. The detection of donor (NADH) and acceptor (R123) fluorescence in different nanosecond time gates following the exciting laser pulses enhances selectivity and improves quantification of energy transfer measurements. Therefore, timegated energy transfer spectroscopy is suggested to be an appropriate tool for probing mitochondrial malfunction.     

应用荧光探针研究菠菜叶绿体膜对低强度电磁场的辐射敏感性

应用ANS渗入菠菜叶绿体膜后的ANS荧光光谱和光合细胞Chla荧光参数的变化研究了菠菜叶绿体膜对辐射功率密度为5 mW·cm-2以下的300 MHz低强度电磁场的辐射敏感性。研究发现,在1～5 mW·cm-2的低强度电磁场作用下,菠菜叶绿体ANS荧光光谱的位置没有明显变化,但ANS荧光强度明显增大,表明低强度电磁场使菠菜叶绿体膜流动性变小。1～5 mW·cm-2低强度电磁场的作用还使菠菜叶绿体发出的Chla荧光参数F₀减小,f_V／F₀,F_V／F_m和ΔF_V／T增大,F_VI／F_V减小,表明低强度电磁场使菠菜叶绿体膜发生了光系统Ⅱ(PSⅡ)无活性中心向有活性中心的转变,PSⅡ潜在活性提高、光合电子传递过程加快,原初光能转换效率增强。菠菜叶绿体膜ANS荧光和Chla荧光对低强度电磁场的这种辐射敏感性说明了低强度电磁场能对菠菜光合作用系统产生非热效应,并且,菠菜光合细胞有可能通过PSⅡ活性中心异质性的转变来适应电磁辐射增强的环境。     

Specific molecular properties of organic solvents determine the fluorescence depolarization of DPH and TMA-DPH in membranes

In pig liver microsomes and protein-free egg PC liposomes the effects of organic solvent molecules on the fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylamino)phenyl]-6-phenyl-hexa-3,5-triene (TMA-DPH) were investigated. Aromaticity, alkyl chain length, and stereometry of the solvent molecules are shown to determine the changes of fluorescence depolarization. A concentration-dependent decrease in the fluorescence anisotropy is obtained with aromatic molecules but not with aliphatic molecules. The decrease correlates with the increasing side chain length of alkylbenzenes for both fluorophors and both membrane systems. Benzene in microsomes deviates characteristically from this trend. Measurements of TMA-DPH reveal smaller changes of anisotropy but yield the same qualitative results. Moreover, it is possible to distinguish the effects of the different stereometric properties of the three xylene isomers on the fluorescence anisotropy of DPH. According to their alkyl chain length and their specific stereometry, they exert the strongest fluidizing effect of all solvents investigated.     

Fluorescence lifetimes of diphenylhexatriene-containing probes reflect local probe concentrations: Application to the measurement of membrane fusion

An important process in the life of a cell is fusion between cellular membranes. This is the process by which two cellular compartments surrounded by different membranes join to become a single compartment surrounded by a single membrane, without significant loss of compartment contents. To demonstrate fusion, the cell biophysicist must demonstrate all three critical aspects of the process: (1) mixing of membrane components, (2) mixing of compartment contents; and (3) retention of compartment contents. Most commonly, accomplishing this involves the use of fluorescence probes. The general theme to the methods described involves some form of concentration-dependent quenching. An unique method developed in our laboratory utilizes the concentration dependence of the fluorescence lifetime of a phosphatidylcholine containing carboxyethyl diphenylhexatriene at position 2 and palmitic acid at position 1 of glycerol (DPHpPC). The fluorescence lifetime of this molecule and that of its parent fluorophore diphenylhexatriene (DPH) shorten dramatically as their two-dimensional concentrations in a membrane increase. This lifetime quenching can be described by dimer formation that reduces the symmetry of the DPH excited state. This phenomenon allows one to use the fluorescence lifetime to gain insight into the local concentration of probe in microscopic regions of a membrane. One application of this is in distinguishing lipid transfer between the outer leaflets of two contacting membrane bilayers from fusion between these membranes that leads to mixing of lipids in both the inner and outer leaflets of the membrane bilayers. This allows a single measurement to demonstrate fusion between membrane pairs.Abbreviations PEG poly(ethylene glycol) - Na₂EDTA ethyiene-diamine-tetraacedic acid, disodium salt - LUV large, unilamellar vesicles made by rapid extrusion technique - DPH 1,6-diphenyl-trans-1,3,5-hexatriene - DPHpPC 1-palmitoyl-2-[[[2-[4- (phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]oxy]carbonyl]-3-sn-phosphatidylcholine - DPPC 1,2-dipalmitoyl-3-sn-phosphatidylcholine - PA palmitic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE - Rh-PE N-(lissamine Rhodamine B sulfoyl)-PE - R₁₈ octadecyl Rhodamine B chloride - ANTS 1-aminonaphthalene-3,6,8-trisulfonic acid - DPX N,N-p-xylylene-bis(pyradinium bromide)     

DNA Vector Polyethyleneimine Affects Cell pH and Membrane Potential: A Time-Resolved Fluorescence Microscopy Study

Polyethyleneimine (PEI) is one of the very efficient nonviral vectors being developed and tested for artificial gene transfer into target cells. One of its serious limitations is the significant cytotoxicity of the large amounts of free PEI in the mixtures of DNA and PEI used for transfection. To further investigate the cellular effects of free PEI, we have analyzed the PEI-induced alterations of various cell parameters such as membrane heterogeneity and fluidity, cytoplasmic pH, and plasma membrane potential in a variety of cells such as Swiss 3T3 fibroblast, Chinese hamster ovary, insect cells SF9, plant cell line BY2, and Saccharomyces cerevisae. Fluorescence probes such as Nile red, SNARF-1, and cyanine dye DiSC₂(3), coupled with the technique of picosecond time-resolved fluorescence microscopy, were used in estimating the above-mentioned cell parameters. It was found that the cell membranes were largely unperturbed by PEI. However, the cytoplasmic pH showed an increase of 0.1–0.4 units when the cells were treated with PEI. The plasma membrane potential was found to be depolarized in S. cerevisae and Swiss 3T3 cells. These results suggest that the cytotoxic effects of PEI may partly originate from inhibition of regulation of cytoplasmic pH and plasma membrane potential. Further, it is proposed that the resultant cell alterations favors the transfection process.     

Investigation of the Dynamic Properties of the Lipid Layer of Intraplastid Membranes by Lipophilic Fluorescent Probes

The dynamic properties of the lipid layer of intraplastid membranes have been studied by analyzing the stationary and kinetic spectral polarization characteristics of the fluorescence of lipophilic probes of pyrene and diphenylhexatriene (DPH). Based on the data of the decay of the fluorescence anisotropy of pyrene, the value of the microviscosity of lipids in the membranes of prolamellar bodies (PLB) and protilakoids of etioplasts has been calculated. The pyrene molecules built into the membranes of etioplasts have a relatively high rotational mobility (stationary anisotropy r _s < 0.1). The DPH molecules rotate with difficulty in etioplast membranes (r _s > 0.3). After photoreduction of protochlorophyllide (Pd) in vivo, the rotation of the pyrene and DPH molecules in the membranes of prolamellar bodies becomes easier and this leads to a decrease in r _s. Illumination raised the degree of excimerization of the pyrene immersed into lipids (_exc = 337 nm), and the microsurrounding of the molecules of the probe in lipids became more hydropholic (the relationship between the vibronic maxima at 373 and 387 nm decrease). The set of data obtained points to a decrease in the microviscosity of the lipid layer of the membranes of prolamellar bodies as a result of illumination of sproutings.     

The Susceptibility of Non-UV Fluorescent Membrane Dyes to Dynamical Properties of Lipid Membranes

Fluorescence spectroscopy and microscopy are powerful techniques to detect dynamic properties in artificial and natural lipid membrane systems. Unfortunately, most fluorescent dyes that sense dynamically relevant membrane parameters are UV sensitive. Their major disadvantage is a high susceptibility to fluorescence bleaching. Additionally, the risk for hazardous damages in biological components generally increases with decreasing excitation wavelength. Therefore the use of non-UV–sensitive membrane dyes would provide significant advantage, particularly for applications in fluorescence microscopy, which usually implies high local excitation intensities. We applied steady-state fluorescence spectroscopy techniques to several UV and non-UV membrane dyes to detect and compare dynamically relevant excitation and emission characteristics. Small unilamellar liposomes (composed of egg yolk phosphatidylcholine) served as a model system for biological membranes. The dynamic properties of the membranes were varied by two independent parameters: the intrinsic cholesterol content (0–50 mol%) and temperature (10–50°C). We tested four non-UV–sensitive membrane dyes: 9-diethylamino-5H-benzophenoxazine-5-one (Nile Red), 4-(dicyanovinyl)julolidine (DCVJ), N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM 4-64), and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiIC₁₈). We also tested three derivatives of DiIC₁₈: DiIC₁₆ and DiIC₁₂ differ in acyl chain length and Fast-DiIC₁₈ provides double bonds between hydrocarbon atoms. The spectral results were compared to established fluorescence characteristics of four UV membrane dyes: the anisotropy of 1-6-phenyl-1,3,5,-hexatrien (DPH), two derivatives of DPH (TMA-DPH and COO^–-DHP), and the generalized polarization of 6-dodecanoyl-2-dimethyl-aminonaphthalene (Laurdan). Our results indicate that the tested non-UV dyes do not reveal dynamically relevant membrane parameters in a direct manner. However, spectral characteristics make DiIC₁₈, Nile Red, and DCVJ promising probes for the microscopic detection of lateral lipid organization, an indirect indicator of membrane dynamics. In particular, DiIC₁₈ showed very selective shifts in the emission spectra at defined temperatures and cholesterol contents that have not been reported elsewhere.     

荧光偏振光谱法探测光动力过程中癌细胞膜的流动性

以DPH作荧光探剂,用荧光偏振光谱法研究了金属酞菁配合物苯硫基钛菁锌(C₅₆H₃₂N₈S₄Zn)在光动力过程中,不同光照时间对乳腺癌细胞膜流动性的影响。实验结果表明,经光照激发光敏剂金属酞菁后,荧光标记团的偏振度增大,癌细胞膜流动性降低,微黏度升高。说明这是光动力治疗的疗效所在。用酶联免疫检测(MTT法)不同光照时间对细胞存活的影响,结果显示癌细胞最低存活率与膜流动性降低成正相关。这表明光动力过程在一个相近的程度影响癌细胞膜的流动性和增殖,提示光动力过程可能通过影响细胞膜流动性引起细胞增殖变化,即细胞膜是光动力作用的一个位点。     

铝离子对辅酶NADH分子荧光增强和构象变化的影响

烟酰胺腺嘌呤二核苷酸(NADH)是生物体内重要的辅酶分子,在细胞能量代谢中发挥着关键作用。金属离子可以影响NADH所参与的酶促反应,其中铝离子（Al3+）对神经系统具有毒性,可以引发神经退行性疾病。因此,Al3+和NADH分子间相互作用的研究有助于了解Al3+对生物体内TCA循环和酶促反应的影响,具有重要的生物学意义。本文采用紫外-可见吸收和稳态荧光光谱,结合时间相关单光子计数技术(TCSPC),研究了Al3+对水溶液中NADH的本征荧光光谱和分子构象变化的影响。紫外-可见吸收光谱显示,NADH与Al3+的结合不会改变NADH分子腺嘌呤和烟酰胺两个本征发色团的吸收特性。为避免NADH分子内两个本征发色团之间的荧光共振能量转移效应的影响,采用340 nm作为激发波长,比较了NADH与Al3+作用前后的荧光特性。实验结果证实,Al3+可以与NADH焦磷酸盐桥上的两个氧原子相结合,使NADH分子的结构变得相对更加刚性,从而抑制NADH分子在溶液中的转动等非辐射过程,导致NADH分子平均荧光寿命增加,最终引起NADH分子荧光强度随Al3+浓度的增加而线性增强。进一步,采用NADH本征荧光寿命振幅比的研究方法表征了NADH分子在溶液中的两种主要构象形式：腺嘌呤和烟酰胺相互堆积的折叠构象以及腺嘌呤和烟酰胺相互分离的展开构象。研究发现,Al3+会打破溶液中NADH分子展开构象和折叠构象的平衡状态,促使辅酶NADH分子的展开构象转变为折叠构象,最终达到新的动态平衡,并且当NADH和Al3+以不大于1∶2的浓度比结合时,NADH分子两种构象的振幅比与铝离子浓度的对数间存在线性关系,在Al3+浓度检测等领域具有良好的应用前景。     

Spatially resolved simulations of membrane reactions and dynamics: Multipolar reaction DPD

Biophysical chemistry of mesoscale systems and quantitative modeling in systems biology now require a simulation methodology unifying chemical reaction kinetics with essential collective physics. This will enable the study of the collective dynamics of complex chemical and structural systems in a spatially resolved manner with a combinatorially complex variety of different system constituents. In order to allow a direct link-up with experimental data (e.g. high-throughput fluorescence images) the simulations must be constructed locally, i.e. mesoscale phenomena have to emerge from local composition and interactions that can be extracted from experimental data. Under suitable conditions, the simulation of such local interactions must lead to processes such as vesicle budding, transport of membrane-bounded compartments and protein sorting, all of which result from a sophisticated interplay between chemical and mechanical processes and require the link-up of different length scales. In this work, we show that introducing multipolar interactions between particles in dissipative particle dynamics (DPD) leads to extended membrane structures emerging in a self-organized manner and exhibiting the necessary mechanical stability for transport, correct scaling behavior, and membrane fluidity so as to provide a two-dimensional self-organizing dynamic reaction environment for kinetic studies in the context of cell biology.     

Membrane Fluidity of the Pentachlorophenol-Mineralizing Sphingomonas sp. UG30

The effect of PCP and NaPCP on the cytoplasmic membranes of the PCP-mineralizing bacterium Sphingomonas sp. UG30 was assessed using fluorescence polarization and total cellular fatty acid analysis. Direct exposure of resting UG30 cells to PCP up to 250 ppm and NaPCP up to 1000 ppm did not cause any changes in the polarization ratios or the fatty acid profile of the UG30 cytoplasmic membranes. Growth of UG30 cells in the presence of 25 ppm NaPCP did not affect the total cellular fatty acid profile or membrane fluidity as observed by fluorescence polarization.     

Three-photon-induced fluorescence of diphenylhexatriene in solvents and lipid bilayers

We observed the emission of l,6-diphenyl-l,3,5-hexatriene (DPH) when excited with the fundamental output of a fs Ti:sapphire laser at 860 nm. The emission spectra of DPH were identical to that observed for one-photon excitation at 287 nm. The dependence of the DPH emission intensity on laser power was cubic, indicating three-photon excitation of DPH at 860 nm. At a shorter wavelength of 810 nm, the dependence on laser power was quadratic, indicating a two-photon process. At an intermediate wavelength of 830 nm the mode of excitation was a mixture of two- and three-photon excitation. At 830 nm the anisotropy is no longer a molecular parameter, and the mode of excitation and anisotropy of DPH depends on laser power. Frequency-domain anisotropy decays of DPH in triacetin revealed the same rotational correlation times for two- and three-photon excitation. However, the time 0 anisotropy of DPH was larger for three-photon excitation than for two-photon excitation. Steady-state anisotropy data for DPH-labeled membranes revealed the same transition temperature for one- and three-photon excitation. These anisotropy data indicate that membrane heating was not significant with three-photon excitation and that three-photon excitation may thus be of practical usefulness in fluorescence spectroscopy and microscopy of membranes.     

弹性蛋白酶发酵过程中发酵液紫外吸收光谱的研究

研究了弹性蛋白酶发酵过程中发酵液紫外吸收光谱的变化,分析比较了发酵液吸收光谱的变化与细菌生长以及发酵产酶过程之间的联系.结果表明,弹性蛋白酶发酵液紫外吸收光谱的变化与菌体生长以及弹性蛋白产酶过程存在密切联系.紫外吸收光谱技术可以用于检测发酵液中有机氮源消耗以及弹性蛋白酶的产生等动态变化,研究为检测弹性蛋白酶发酵的动态过程提供一种比较新颖的方法,为建立紫外光谱技术在线检测发酵过程奠定了基础.