Photobleaching Method Research Articles

Involvement of connexin 43 in human trophoblast cell fusion and differentiation.

The syncytiotrophoblast is the principal component of the human placenta involved in feto-maternal exchanges and hormone secretion. The syncytiotrophoblast arises from the fusion of villous cytotrophoblasts. We recently showed that functional gap junctional intercellular communication (GJIC) is an important prerequisite for syncytiotrophoblast formation and that connexin 43 (Cx43) is present in cytotrophoblasts and in the syncytiotrophoblast. To determine whether Cx43 is directly involved in trophoblast fusion, we used an antisense strategy in primary cultures of human villous cytotrophoblasts that spontaneously differentiate into the syncytiotrophoblast by cell fusion. We assessed the morphological and functional differentiation of trophoblasts by desmoplakin immunostaining, by quantifying hCG (human chorionic gonadotropin) production and by measuring the expression of specific trophoblast genes (hCG and HERV-W). Furthermore, we used the gap-FRAP (fluorescence recovery after photobleaching) method to investigate functional GJIC. Cytotrophoblasts treated with Cx43 antisense aggregated and fused poorly. Furthermore, less HERV-W env mRNA, hCGbeta mRNA and hCG secretion were detected in Cx43 antisense-treated cytotrophoblasts than in cells treated with scrambled antisense. Treatment with Cx43 antisense dramatically reduced the percentage of coupled trophoblast cells. Taken together, these results suggest that Cx43 is directly involved in human trophoblast cell-cell communication, fusion and differentiation.

Experimental studies of electroosmotic flow dynamics in microfabricated devices during current monitoring experiments.

Electroosmotic flow (EOF) was monitored in glass microfluidic devices at rates up to 2 Hz with a precision of 0.2-1.0% using a technique based on the periodic photobleaching of a dilute, neutral fluorophore added to the running buffer. This EOF monitoring method was used to examine the performance of the current monitoring technique for measuring an average electroosmotic flow in a microfluidic device with a cross-T design. Flow measurements made with the current monitoring method gave a precision of 0.4-2.2%, but the periodic photobleaching method shows that the current monitoring technique causes changes in EOF as high as 41% during a single experiment. The periodic photobleaching method for EOF monitoring was also used to study EOF in channels on opposite sides of a cross-channel intersection. The opposite channels were shown to exhibit substantially different EOF dynamics during a current monitoring experiment as well as different steady-state EOF rates during normal operating conditions.

Connexin expression and gap junctional intercellular communication in human first trimester trophoblast.

Connexin (Cx) expression and gap junctional intercellular communication (GJIC) are involved in development and differentiation processes, and recently mutation of connexin genes has been implicated in pathologies. In the human placenta, two distinct differentiation pathways of cytotrophoblastic cells coexist and lead to a fusion phenotype (villous trophoblast) and a proliferative/invasive phenotype (extravillous trophoblast). Here we characterized in situ and in vitro the expression of Cx transcripts and proteins in the villous and extravillous trophoblast of first trimester placenta. In addition, the GJIC functionality was investigated using the gap-fluorescence recovery after photobleaching (gap-FRAP) method. We demonstrated in the villous trophoblast the presence of Cx43 mRNA and of Cx43 protein localized between cytotrophoblastic cells and between cytotrophoblastic cells and syncytiotrophoblast. In vitro, a transient functional gap junctional intertrophoblastic communication was demonstrated during the trophoblast fusion leading to the multinucleated syncytiotrophoblast. During the proliferative process of the extravillous trophoblast, Cx40 is expressed in the proximal part of the cell columns. When cytotrophoblastic cells were cultured on Matrigel for 2 days, alpha5beta1 integrin expression was observed concomitant with the presence of Cx40 mRNA and of Cx40 protein between the cells. No evidence for a GJIC was detected in this induced extravillous phenotype. In addition, Cx32 was detected between some aggregated cells after 72 h of culture. Our data show that the presence of Cx43 allows an inter-trophoblastic GJIC and is associated with the fusion process leading to the villous syncytiotrophoblast and that the presence of Cx40 does not allow GJIC and is associated with the extravillous phenotype.

Comparison of the lateral diffusion coefficient of hepatocyte plasma membrane proteins in three strains of sensescence accelerated mouse (SAM)

The lateral diffusion coefficients of proteins ( D p) were measured in hepatocyte plasma membrane in freshly prepared liver smears by means of the fluorescence recovery after photobleaching (FRAP) method. D p was measured after development of peroxide-induced autofluorescence (PIAF) in a total of 115 senescence accelerated mice (SAM), distributed in three strains, at least five age-groups in each, as follows: (i) SAMR1TA (25 males and 22 females), medium life span (MLS) in months, under specific pathogen free (SPF) conditions, MLS spf 20.1 and 20.0, respectively, while under conventional conditions, MLS Δpf=18.9 in average for both sexes; (ii) SAMP6/Ta (18 males and 17 females), MLS spf=17.1 and 15.3, respectively, and MLS cc=8.1 for both sexes; (iii) SAMP6/Ta (17 males and 16 females), MLS spf=15.6 and 14.7, respectively, and MLS cc=10.0 for both sexes. A highly significant negative linear age-correlation of D p ( R=0.975 or higher) was found in each strain, being roughly proportional with the MLS cc values. Since the studied mice kept under SPF conditions survived longer, than under conventional conditions, the actual age-dependent decay rates of D p values did not differ significantly in two pairs of comparisons (female R1/P6 and female R1/P8), whereas they did in all other possible pairs, including also the normal C57BL/6 mice. The main conclusion can be drawn that the D p of hepatocyte membranes is a good biomarker of aging and survival also in SAM, as in all other inbred and outbred rodents, studied so far.

Lifetime of spatially heterogeneous dynamic domains in polystyrene melts

A photobleaching method is used to measure the rotational dynamics of tetracene and rubrene in polystyrene, and to determine the lifetime of spatially heterogeneous dynamic domains in the polystyrene matrix. It is possible to selectively photobleach subsets of probe molecules in more mobile environments and to measure the time required for the remaining slower-than-average probes to be redistributed into an equilibrium set of environments. For polystyrene, this exchange time is much longer than the α-relaxation time at temperatures very near Tg and exhibits a strong temperature dependence. These results are qualitatively consistent with previous studies on tetracene in supercooled o-terphenyl and indicate that the observation of long-lived heterogeneous dynamic domains at temperatures very near Tg is neither matrix nor probe specific.

Optical Patterning in Organic EL Devices

Fabrication of micropatterned multicolor organic electroluminescent (EL) devices by the “photobleaching method” is described. In this approach, we take advantage of the instability of organic fluorescent molecules upon photooxidation; that is, organic molecules can be oxidized or bleached by exposure to light in the presence of oxygen. Several types of multicolor devices having a dye-dispersed polymer emitter layer were fabricated. In order to tune the emission color and pattern the device, the polymer emitter layer is exposed to ultraviolet light or visible light after formed by spin coating so that specific dyes are photooxidized and become non-emissive. Due to this photobleaching process, the light-exposed area have different emission colors from the non-exposed area, and, using a fine-patterned photomask, micropatterned multicolor EL devices were successfully fabricated. This photobleaching method is the simplest method for the fabrication of multicolor polymer EL devices and can be applicable to the fabrication of full color displays having arrays of red-green-blue pixels.

Molecular motion during physical aging in polystyrene: Investigation using probe reorientation

A photobleaching method has been used to observe the reorientation of tetracene and rubrene in polystyrene during physical aging. Rotation times change more than an order of magnitude during isothermal aging after a temperature quench from above Tg. Down- and up-jumps of the temperature show the expected asymmetry due to the nonlinearity of the aging process. The rotation times of tetracene and rubrene require the same amount of time to reach equilibrium after a temperature change (103 − 105 s in the range 93–99 °C). These equilibration times are the same order of magnitude as equilibration times for volume and enthalpy relaxation, but have a somewhat weaker temperature dependence. Very near equilibrium, the rates of aging are different for the two probes, with rubrene approaching equilibrium more rapidly at very long times. This may be understood if the aging process is spatially heterogeneous, that is, if aging occurs more rapidly in some small regions of the sample than in others. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 68–79, 2000

Hypoxia-reoxygenation inhibits gap junctional communication in cultured human umbilical vein endothelial cells.

We studied the change in gap junctional intercellular communication (GJIC) on human umbilical vein endothelial cells (HUVEC) under hypoxia-reoxygenation (H-R) conditions by the fluorescence redistribution after photobleaching (FRAP) method. Confluent HUVEC monolayers were exposed to hypoxia (pO2<0.1%) for 12 hours, and then were returned to normal atmospheric conditions for reoxygenation. Contrast microscopic observation showed no significant changes in the morphology of the HUVEC at any times after H-R. Reoxygenation following hypoxia caused time-dependent decrease in GJIC, that is, GJIC reduction was induced after 2 hours and reached maximum at 4-6 hours which recovered to normal levels after 18 hours. Oxidant sensitive fluorescence dye assay revealed that the generation of intracellular free radicals increased during the first 2 hours after reoxygenation. Hydroxyl radical scavengers (MCI-186, DMSO) and an iron chelator (deferoxamine) abolished the reduction of GJIC due to H-R. However, SOD, catalase and probucol were essentially inactive on this reduction. These data suggest that ischemia-reperfusion injury may be caused by a functional defect of GJIC induced by reactive oxygen radicals.

Molecular motion during physical aging in polystyrene: Investigation using probe reorientation

A photobleaching method has been used to observe the reorientation of tetracene and rubrene in polystyrene during physical aging. Rotation times change more than an order of magnitude during isothermal aging after a temperature quench from above Tg. Down- and up-jumps of the temperature show the expected asymmetry due to the nonlinearity of the aging process. The rotation times of tetracene and rubrene require the same amount of time to reach equilibrium after a temperature change (103 − 105 s in the range 93–99 °C). These equilibration times are the same order of magnitude as equilibration times for volume and enthalpy relaxation, but have a somewhat weaker temperature dependence. Very near equilibrium, the rates of aging are different for the two probes, with rubrene approaching equilibrium more rapidly at very long times. This may be understood if the aging process is spatially heterogeneous, that is, if aging occurs more rapidly in some small regions of the sample than in others. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 68–79, 2000

Highly Efficient Organic/Inorganic Hybrid Nonlinear Optic Materials via Sol−Gel Process: Synthesis, Optical Properties, and Photobleaching for Channel Waveguides

The thermally stable second-order nonlinear optic (NLO) hybrid materials were successfully prepared by a sol−gel process using dye-attached sol−gel monomers (ormosils) of difunctionalized or trifunctionalized second-order NLO chromophores attached to silicon atoms. The dye-attached sol−gel monomers were synthesized by the coupling reaction of 3-isocyanatopropyltriethoxysilane with the corresponding functionalized NLO chromophores in DMAc. The dye-attached sol−gel monomers were homopolymerized and copolymerized with TEOS by hydrolysis and polycondensation in the presence of a slight excess amount of acidified water (pH 3), yielding the hybrid precusors (or a homogeneous solution). The hybrid precursors were spin-coated on various substrates such as ITO glass and NaCl disk. Hybrid films were further polycondensed by curing at 170 °C for 3 h. The cured hybrid materials existed in monohydroxy (T2, Q3) and nonhydroxy (T3, Q4) states, indicating the formation of a highly cross-linked silicon−oxygen polymeric network. All of the cured organic/SiO2 hybrid films are transparent. The organic/SiO2 hybrid materials were quite stable at elevated temperatures above 250 °C. The hybrid films were poled by corona discharge technique or dc contact electrode. The values of electro-optic coefficient for the organic/SiO2 hybrid materials were in the range of 3.7 to 16 pm/V at the wavelength of 1.3 μm, depending on dye-attached sol−gel monomers and poling conditions. The SG-DANS (1:2:2) film shows excellent temporal stability. The r33 signal remained at 80% of its initial value after heating even at 150 °C for 3 h. Also, the second-order nonlinear optical coefficient d31 of 72 pm/V for the SG-DANS (1:2:0) hybrid film measured by the Maker fringe technique was obtained. The refractive index was decreased after photobleaching. Using the hybrid film, a single-mode channel waveguide was also fabricated by the photobleaching method. The mode pattern confirmed that a channel waveguide was single-mode, and the optical propagation loss was measured to be less than 1 dB/cm.

Age-dependence of the lateral diffusion coefficient of lipids and proteins in the hepatocyte plasma membrane of BN/BiRijHsd rats as revealed by the smear-FRAP technique

The lateral diffusion coefficients of lipids ( D l) and proteins ( D p) were measured in hepatocyte plasma membranes in freshly prepared liver smears of Brown Norway (BN/BiRijHsd) rats by means of the fluorescence recovery after photobleaching (FRAP) method. D l was measured after staining with N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE) in 14 males in four age groups (4.3–30.4 months), and 12 females (5.0–31.0 months). D p was measured after the development of peroxide-induced autofluorescence (PIAF) in 16 males in four age groups (4.7–30.8 months) and 15 females in four age groups (4.0–28.1 months). Highly significant negative, linear age-correlations of D l (R=−0.972 in males, and −0.840 in females, respectively) were found, the average data being 1.39×10 −9 cm 2/s in the young male rats, and only 9.26×10 −10 cm 2/s in the oldest ones, whereas in females, these two extremes were 1.44×10 −9 and 8.97×10 −10 cm 2/s, respectively. Protein lateral mobility also declined linearly with age; in the male rats it decreased from 2.7×10 −10 to 1.8.×10 −10 cm 2/s (R=−0.903), while in the females it decreased from 2.64×10 −10 to 2.21×10 −10 cm 2/s (R=−0.955) between the youngest and oldest age groups. There were also some characteristic differences in the fractional recoveries (FR%), too. These trends are comparable with those of F344 rats studied previously, supporting the conclusion that the changes in the lateral mobility of plasma membrane proteins and lipids represent important biomarkers of cellular aging also in the BN/BiRijHsd rats.

Down-regulation of gap junctional intercellular communication between osteoblastic MC3T3-E1 cells by basic fibroblast growth factor and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate).

To address the relation between osteoblast growth and cell-to-cell communication, we examined the effects of basic fibroblast growth factor (bFGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), both potent stimulators of osteoblastic proliferation, on gap junctional intercellular communication between osteoblastic MC3T3-E1 cells. The level of intercellular communication was estimated by a photobleaching method. TPA inhibited the degree of intercellular communication in two different time-dependent manners. The early (< 1 h) inhibition by TPA was consistent with an increase in the phosphorylation of connexin 43 (Cx43). The later inhibition was caused by reduction in the total amount of Cx43 on the plasma membrane, due to the decrease in the level of Cx43 transcripts. These qualitative and quantitative modulations by TPA were inhibited by a selective inhibitor of protein kinase C, GF109203X. bFGF also attenuated the gap junctional intercellular communication. However, short exposure (< 5 h) to bFGF did not affect the communication. The fact that the growth factor immediately stimulated the phosphorylation of Cx43 indicates that the phosphorylation site(s) affected by bFGF was not involved in the inhibition of communication. The decrease in the intercellular communication level was detected by the longer exposure (> 8 h) to bFGF and paralleled the decline in the Cx-mRNA level. This inhibitory effect of bFGF was abolished by the addition of a tyrosine kinase inhibitor, herbimycin A. Thus, gap junctional intercellular communication between osteoblasts was down-regulated by osteoblastic mitogens through different mechanisms of the modulation of Cx43.

Photobleaching of polydiacetylene waveguides: a characterization of the process and patterning of optical elements

The photobleaching process of poly-3butoxyl-carbonyl-methyl-urethane (poly-3BCMU) waveguides by means of an UV lamp and the 488-nm line of an Ar laser is characterized and modeled. The limits of the theory are discussed in light of experimental results, and we stress the role of the oxygen diffusion rate on the process. Finally, we adopt the photobleaching method to pattern a guided-wave micro-optic device and holographic diffraction gratings on spun poly-3BCMU films.

The effect of ethinyl-estradiol treatment on the lateral mobility of lipids and proteins in hepatocyte plasma membrane of male rats (FRAP studies on liver smears)

The lateral diffusion constants of lipids (D 1) and proteins (D p) in the plasma membranes of hepatocytes were measured in liver smears of male Fischer 344 rats, by means of the fluorescence recovery after photobleaching (FRAP) method, after ethinyl-estradiol (EE) treatment of 1 or 5 days. The main finding was that EE treatment results in significant decreases in both the D 1- and the D p-values. These observations are interpreted in terms of the role of membrane physicochemistry in functions of the hepatocytes.

Age-dependence of the lateral mobility of lipids in hepatocyte plasma membrane of male rats and the effect of life-long dietary restriction.

The lateral diffusion constant of lipids (D(1)) in hepatocyte plasma membranes was measured in liver smears by means of the fluorescence recovery after photobleaching (FRAP) method, applying the label, N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE). Nineteen ad libitum fed, male Fischer-344 rats in four age groups (2.1-29.8 months of age) were studied. A highly significant negative linear age-correlation of D(1) (cc = 0.958) was found. D(1) values were 1.39 x 10(-9) cm2/s in the young rats, and only 6.77 x 10(-10) cm2/s in the oldest rats. Lipid lateral mobility is changing in parallel with that of proteins, having been measured previously also with the FRAP method by the authors. Fractional recovery values (FR%) of the lipids were lower than those of proteins even in the young ages, but also decreased linearly with age, therefore, the parameter, D, x FR decreased even steeper with age than D(1) itself. D(1) was also measured in a group of six male Fischer 344 rats having been kept on dietary restriction (DR) since their age of 1 month until 30 months of age (applying the every-other-day (EOD) feeding). DR caused an increase of D(1), compared with the age-matched ad libitum fed animals: the mean was 9.24 x 10(-10) cm(2)/s. FR% and D(I), x FR again increased considerably under DR. The results are interpreted in terms of the increased protein and lipid turnover under DR.

Human granulosa cells in culture exhibit functional cyclic AMP-regulated gap junctions.

Numerous gap junctions exist between granulosa cells, between cumulus cells and between cumulus cells and the oocyte. They may play a role in the regulation of both follicular development and oocyte status. We used primary cultures of human granulosa cells to study the molecular nature and functionality of these gap junctions. As shown by a cinemicrographic technique, during the first 3 days of culture, cells flattened and extended in several directions by means of cytoplasmic extensions. An ultrastructural study showed the presence of both intercellular and annular gap junctions after 48 h of culture. As revealed by immunodetection analyses, connexin 43 was present. An analysis using a functional procedure, the gap fluorescence recovery after photobleaching (FRAP) method, indicated that: (i) diffusional communication existed among granulosa cells; (ii) the communication was delayed by treatment with 1-heptanol, a well-documented inhibitor of gap junction permeability; and (iii) permeability was up-regulated by incubation with 8-Br-cAMP, an analogue of cyclic AMP. The detection of connexin 43 and functional gap junctions in networks of cytoplasmic extensions indicated junction formation among cells during culture. In conclusion, our results show that human granulosa cells in culture exhibited functional gap junctions. Connexin 43 was present and the permeability of the gap junctions was up-regulated by cyclic AMP, an important modulator of human granulosa cell function.

Probe rotation near and below the glass transition temperature: Relationship to viscoelasticity and physical aging

AbstractReorientation times ťc for two probes in several amorphous polymers near the glass transition temperature Tg are reported. Tg for these polymers ranges from 205 to 459 K. Probe reorientation was measured in the time window from 10−2 to 104 s with a recently developed photobleaching method. ťc for a given probe at the Tgs of the different polymers varies more than three decades. Viscoelastic relaxation times characteristic of the Rouse modes of the matrix polymers are closely related to probe rotation times and thus also not constant at Tg. The characteristic length scale of motions responsible for the glass transition varies significantly for the three polymers studied. Preliminary physical aging results indicate that probe reorientation in polystyrene ages slightly faster than the volume.

Tubulin dynamics in neuronal axons of living zebrafish embryos

The mechanism of cytoskeletal protein transport, especially the question of what kind of form the cytoskeletal proteins assume during transport in neurons in situ, has been an important, as yet unsettled issue. To clear up this matter, we adopted the embryonic zebrafish as a living animal model and applied the fluorescence recovery after photobleaching (FRAP) method. The zebrafish embryo is appropriate for this kind of study because of its transparency during the early developmental stage, allowing the observation of neurons that incorporate the microinjected fluorescent tubulin directly under fluorescence microscopy. FRAP revealed no movement of the bleached zone proximodistally, where fluorescence recovered gradually (recovery half-time, 44.2 +/- 11.2 min; n = 36), suggesting that the polymers are stationary but dynamic and that the true moving form could be small oligomers or heterodimers.

Molecular Motions and Viscoelasticity of Amorphous Polymers near Tg

Rotational correlation times τ c for rubrene and tetracene are reported near and below T g in three polymers: polyisobutylene, polystyrene, and Bisphenol A polysulfone. A photobleaching method was used to obtain τ c values from 10 -1 to 10 3 s. In each polymer matrix, the orientation autocorrelation function for tetracene (the smaller probe) decays more rapidly and less exponentially than the correlation function for rubrene. τ c for a given probe at the T g of the matrices varies more than 3 decades. For the three polymers studied, probe rotation times at T g showed a systematic decrease with increasing matrix T g . Viscoelastic relaxation times characteristic of the Rouse modes of the matrix polymers are closely related to probe rotation times and also not constant at T g . Thus T g is not an isolocal mobility state for molecular motions on a fixed length scale. On the other hand, the viscoelastic relaxation time associated with the glassy modulus is almost constant at T g . These results suggest that the characteristic length scale for motions associated with the relaxation of the glassy modulus varies significantly for the three polymers studied. Trends in the KWW β values which describe probe reorientation support this interpretation

Lateral mobility of phospholipid molecules in thin liquid films

The Fluorescence Recovery After Photobleaching (FRAP) method was applied to measure the lateral mobility of the fluorescent lipid analog, dioctadecylindocarbocyanine perchlorate (Dil-C18), in microscopic thin liquid films (Foam Films (FFs)). The foam film structures were comprised of two phosphatidylcholine monolayers adsorbed at air/water interfaces which sandwiched a thin liquid core. Lateral diffusion of the DiI molecules in the plane of the monolayers was determined as a function of the thickness of the thin liquid core of the film between the FF monolayers. The results obtained indicated that the diffusion coefficient was strongly dependent both on the distance between the FF monolayers in the range 4 nm to 85 nm (corresponding to the FF thickness) and on the film type. The applicability of the FRAP method for studying the molecular mobility in phospholipid FFs was demonstrated. Considerable differences in the surface diffusion coefficient of Dil were observed, ranging between 2 × 10−8 cm2/s and 22 × 10−8 cm2/s in so called yellow, gray, common black and Newton black FFs. The effect of the presence of polyethylene glycol (PEG-400) in the liquid core of lecithin FFs on surface diffusion was also studied. The surface diffusion results from the FF studies were compared with data from black lipid membranes (BLMs). These structures are related in thickness terms but the molecular orientation in FFs is the reverse of that in BLMs.