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First observation of fluorescence self-quenching in Langmuir films
Volume 193, number 5
CHEMICAL PHYSICS LETTERS
5 June 1992
First observation of fluorescence self-quenching in Langmuir films George R. Ivanov Laboratory of Biomolecular Layers, Institute of Solid State Physics, Bulgarian Academy of Sciences, 72 Trakia boulevard, 1784 SoJia, Bulgaria
Received 9 March 1992
Systematic investigations of dipalmitoyl-phosphatidylethanolamine head labelled with nitrobenzoxadiazole (DPPE-NBD) are performed both on monolayers at the air-water interface and deposited on solid substrates as Langmuir-Blodgett films. This dye compound is frequently used as a fluorescent additive in fluorescence microscopy investigations. It is also a suitable model compound. We present experimental evidence for the self-quenching of the fluorescence when higher-order domains of the dye are formed at the air-water interface. A coexistence of two completely different types of domains at the phase coexistence region for a single-component system is observed. From small-angle X-ray diffraction, pressure-area isotherms and molecular space filling models, an average tail tilt angle of 31’ towards the normal to the substrate surface is estimated when deposition is carried out at 50 mN/m. These data, together with information from polarized FTIR spectra, support a conclusion of a decrease of tail tilt and increase of the order of the deposited films on increase of the deposition pressure.
1. Introduction The fluorescence microscopy technique for investigation of Langmuir and Langmuir-Blodgett (LB) films was introduced in the early 1980s. With its help phase transitions and their nature, defect structure of crystalline phases, the influence of external electric fields, the shape and size of various domains and their interaction were studied. In most cases when phospholipid domains were investigated dipalmitoyl-phosphatidylethanolamine head labelled with nitrobenzoxadiazole (DPPE-NBD) was used as a staining dye. However in the interpretation of the origin of contrast in these experiments the only mechanism mentioned is the reduced solubility of the dye in the higher-order phase [ 11. Another mechanism was pointed out for a dye with the same chromophoric group: quenching of the NBD fluorescence in those molecular conformations when it is in contact with water [ 2,3]. We present here experimental evidence of a third mechanism: selfquenching of the fluorescence when higher-order doCorrespondence to: G.R. Ivanov, Laboratory of Biomolecular Layers, Institute of Solid State Physics, Bulgarian Academy of Sciences, 72 Trakia boulevard, 1784 Sofia, Bulgaria.
mains of the dye are formed at the air-water interface.
2. Materials and methods DPPE-NBD (Sigma) was supplied in chloroform solution. The claimed purity was above 98% and it was used without further purification. Mill&Q liltered water was used. For spectroscopic measurements in the UV and VIS region, LB films were deposited either on microscope slides or quartz substrates. For small-angle X-ray diffraction and FTIR spectroscopy, films were deposited on silicone wafers. For better cleaning a small trough machined from bulk teflon was used [ 4,5 1. The accuracy of the area measurement was above 0.001 nm2/molecule and the precision of surface pressure measurement was above 0.2 mN/m. For the epifluorescence microscopy, mercury lamp illumination and video recording of the image-processed signal from a SIT TV camera were used. The X-ray diffraction patterns were obtained on the AMUR-K (Institute of Crystallography, Moscow) small-angle diffractometer. Nickel-filtered Cu Ku radiation was used (wavelength 0.154 nm). The
0009-2614/92/$ 05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved.
323
Volume 193, number
CHEMICAL
5
PHYSICS
angular resolution of the detector was 0.02’. The absorption and fluorescence spectra of DPPE-NBD on glass substrates and in vesicle solution were measured with Perkin-Elmer spectrophotometers. The polarized FTIR transmission spectra were taken on a Bruker 113~ at 4 cm-’ resolution.
3. Results and discussion The surface pressure-mean molecular area isotherm of DPPE-NBD at room temperature is shown on fig. 1. The circular gas domains observed by fluorescence microscopy at large areas per molecule liquefy at a pressure of around 0.2 mN/m and area around 2.2 nm2/molecule (fig. 2a). This point of surface pressure rise is approximately three times larger in area per molecule than in other phospholipids which is due to a bulkier and more hydrophilic head. This correlates well with the isotherm of the same compound with shorter tail [ 61. On reaching the break of the isotherm at 8.3 mN/m, small bright spots appear which in ref. [ 2 ] are interpreted as seeds of the new phase. However, we also observe at pressures just above this phase transition large (several tens of micrometers) bright circular domains (fig. 2b). Interestingly, both of these bright domains disappear on increasing the pressure. Together with the bright domains, above this phase
-51 0.0
I
I
I
0.5
1.0
1.5
AREA /MOLECULE
,
2.0 [nm’l
1
2.5
Fig. 1. Surface pressure-mean molecular area isotherm of DPPENBD at 20°C. On the insert is shown the small-angle X-ray diffraction for a LB film consisting of 21 monolayers deposited at a pressure of SO mN/m.
324
LETTERS
5 June 1992
transition small dark crystallization centers appear (fig. 2b). On increasing the surface pressure, these centers grow up like circular higher-order domains (fig. 2~). On further increase of the pressure these domains grow like dendrites and a second population of crystallization centers appear (fig. 2d). At the next phase transition around 26 mN/m the higherorder domains overcome the energetic barrier and start to merge (fig. 2e). This is the first time such a coexistence of two different types of domains as on fig. 2b has been observed. The bright domains can be interpreted either as several monolayers piling up over another or as domains in which the NBD group is projecting out of the water, thus greatly increasing its fluorescence. The second proposal is more probable because the conformation with the NBD group projecting out of the water is with a larger area per molecule and consequently on increase of the surface pressure it should disappear. Qualitatively, the textures in figs. 2b, 2c and 2d can be understood as an interplay of growth kinetics, line energy of the fluid/ordered interface and longrange electrostatic repulsion [ 7 1. For example the change from circular to a dendritic shape of the higher-order domains (fig. 2d) is due to the increased long-range repulsive electrostatic forces within the domain on increase of its area, as the kinetic factors should not be taken into account because of the slow equilibrium compression. The only possible mechanism of contrast in these observations can be the self-quenching of fluorescence when molecules are closely packed. This statement is supported by the observation that fluorescence of the LB films from DPPE-NBD has much lower intensity than in chloroform solution (not shown). There is evidence in the literature that for small unilamellar vesicles the clustering at the phase transition liquid-expanded-liquid-condensed reduces the fluorescence intensity more than six times due to self-quenching [ 8 1. It seems that quenching of the fluorescence on interaction of the NBD group with water [2] simply regularly decreases the total fluorescence of this compound as the NBD group is in permanent contact with water with the one possible exception mentioned above. Further information on the dark domains’ structure can be gained from LB films deposited at pres-
Volume 193, number 5
CHEMICAL PHYSICS LETTERS
5 June 1992
Fig. 2. Domain structure of DPPE-NBD at a temperature of 20°C and different surface pressures: (a) liquid-gas coexistence region P=O.2 mN/m, A ~2.2 nmz/mofecuie; (b) coexistence of two different types of domains just above the main phase transition P=8.3; (c) circular ordered domains, P~9.8 mN/m; (d) coexistence of dendritic domains and a second population of ordered domains, P= 18 mN/m; (e) merging of ordered domains just above the second phase transition, P=27 mN/m.
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Volume 193. number 5
CHEMICAL PHYSICS LETTERS
sures above 26 mN/m. Details of transfer at different conditions and the quality of the structures achieved are given elsewhere [ 9 1. From the position of the diffraction maximum (the insert in fig. 1) for a sample transferred at 50 mN/m a bilayer spacing is calculated to be 5.60&O. 1 nm. Reasonable assumptions are that the tails are in the all-trans configuration and no interdigitation of tails exists. For a molecule with fully extended tails oriented normal to the substrate the total length is 3.09 nm [ 91. This, compared to the measured value of 2.80 nm, gives a tail tilt of 3 lo and a projected tails area of 0.45 nm*. This is in good agreement with the measured area per molecule of 0.44 nm2 for 50 mN/m (fig. 1) . Both X-ray (the shift of the diffraction maximum) and FTIR spectroscopy (comparison of intensities of ya(CH3) at 2962 cm-’ and K(CH,) at 2918 cm-i) provide evidence that tail tilt decreases and the order of the structures increases on increasing the pressure for deposition. From the fluorescence microscopy data it is seen that on increasing the pressure above 26 mN/m the liquid phase gradually disappears and correspondingly the order should increase.
Acknowledgement
Help with the measuring techniques is appreciated
326
5 June 1992
from W. Frey (TU, Munich), V. Erochin (Institute of Crystallography, Moscow), S. &stein (Institut fIlr Physikalische Chemie, Mainz) and D. Tsankov (Institute of Organic Chemistry, Sofia). Fluorescence microscopy measurements were carried out in the Laboratory of Professor H. Mohwald (Institut fir Physikalische Chemie, Mainz) following his kind invitation. Many helpful discussions with Professor A.G. Petrov (Institute of Solid State Physics, Sofia) are kindly appreciated. This work was supported under contracts with the National Science Foundation MU-NM-12 and F19.
References [ I] H. Mohwald, Thin Solid Films 159 ( 1988) 1. [2] H. Bercegol, F. Gallet, D. Langevin and J. Meunier, J. Phys. (Paris) 50 (1989) 2277. [ 3 ] P. Muller and F. Gallet, Phys. Rev. Letters 67 ( I99 I ) 1106. [4] G.R. Ivanov, A.T. Todorov and A.G. Petrov, Makromol. Chem. Macromol. Symp. 46 ( 1991) 377. [ 51 G.R. Ivanov, KG. Kostadinov and A.G. Petrov, Thin Solid Films 207 ( 1992), in press. [6] G.R. lvanov and A.G. Petrov, Mol. Ctyst. Liquid Cryst., in press. [7] H. Mohwald, S. Kirstein, H. Haas and M. Fliirsheimer, J. Chim. Phys. 85 (1988) 1009. [8] J.R. Wiener, Biochemistry 24 (1985) 7651. [ 91 G.R. Ivanov and J. Petkova, submitted for publication.
CHEMICAL PHYSICS LETTERS
5 June 1992
First observation of fluorescence self-quenching in Langmuir films George R. Ivanov Laboratory of Biomolecular Layers, Institute of Solid State Physics, Bulgarian Academy of Sciences, 72 Trakia boulevard, 1784 SoJia, Bulgaria
Received 9 March 1992
Systematic investigations of dipalmitoyl-phosphatidylethanolamine head labelled with nitrobenzoxadiazole (DPPE-NBD) are performed both on monolayers at the air-water interface and deposited on solid substrates as Langmuir-Blodgett films. This dye compound is frequently used as a fluorescent additive in fluorescence microscopy investigations. It is also a suitable model compound. We present experimental evidence for the self-quenching of the fluorescence when higher-order domains of the dye are formed at the air-water interface. A coexistence of two completely different types of domains at the phase coexistence region for a single-component system is observed. From small-angle X-ray diffraction, pressure-area isotherms and molecular space filling models, an average tail tilt angle of 31’ towards the normal to the substrate surface is estimated when deposition is carried out at 50 mN/m. These data, together with information from polarized FTIR spectra, support a conclusion of a decrease of tail tilt and increase of the order of the deposited films on increase of the deposition pressure.
1. Introduction The fluorescence microscopy technique for investigation of Langmuir and Langmuir-Blodgett (LB) films was introduced in the early 1980s. With its help phase transitions and their nature, defect structure of crystalline phases, the influence of external electric fields, the shape and size of various domains and their interaction were studied. In most cases when phospholipid domains were investigated dipalmitoyl-phosphatidylethanolamine head labelled with nitrobenzoxadiazole (DPPE-NBD) was used as a staining dye. However in the interpretation of the origin of contrast in these experiments the only mechanism mentioned is the reduced solubility of the dye in the higher-order phase [ 11. Another mechanism was pointed out for a dye with the same chromophoric group: quenching of the NBD fluorescence in those molecular conformations when it is in contact with water [ 2,3]. We present here experimental evidence of a third mechanism: selfquenching of the fluorescence when higher-order doCorrespondence to: G.R. Ivanov, Laboratory of Biomolecular Layers, Institute of Solid State Physics, Bulgarian Academy of Sciences, 72 Trakia boulevard, 1784 Sofia, Bulgaria.
mains of the dye are formed at the air-water interface.
2. Materials and methods DPPE-NBD (Sigma) was supplied in chloroform solution. The claimed purity was above 98% and it was used without further purification. Mill&Q liltered water was used. For spectroscopic measurements in the UV and VIS region, LB films were deposited either on microscope slides or quartz substrates. For small-angle X-ray diffraction and FTIR spectroscopy, films were deposited on silicone wafers. For better cleaning a small trough machined from bulk teflon was used [ 4,5 1. The accuracy of the area measurement was above 0.001 nm2/molecule and the precision of surface pressure measurement was above 0.2 mN/m. For the epifluorescence microscopy, mercury lamp illumination and video recording of the image-processed signal from a SIT TV camera were used. The X-ray diffraction patterns were obtained on the AMUR-K (Institute of Crystallography, Moscow) small-angle diffractometer. Nickel-filtered Cu Ku radiation was used (wavelength 0.154 nm). The
0009-2614/92/$ 05.00 0 1992 Elsevier Science Publishers B.V. All rights reserved.
323
Volume 193, number
CHEMICAL
5
PHYSICS
angular resolution of the detector was 0.02’. The absorption and fluorescence spectra of DPPE-NBD on glass substrates and in vesicle solution were measured with Perkin-Elmer spectrophotometers. The polarized FTIR transmission spectra were taken on a Bruker 113~ at 4 cm-’ resolution.
3. Results and discussion The surface pressure-mean molecular area isotherm of DPPE-NBD at room temperature is shown on fig. 1. The circular gas domains observed by fluorescence microscopy at large areas per molecule liquefy at a pressure of around 0.2 mN/m and area around 2.2 nm2/molecule (fig. 2a). This point of surface pressure rise is approximately three times larger in area per molecule than in other phospholipids which is due to a bulkier and more hydrophilic head. This correlates well with the isotherm of the same compound with shorter tail [ 61. On reaching the break of the isotherm at 8.3 mN/m, small bright spots appear which in ref. [ 2 ] are interpreted as seeds of the new phase. However, we also observe at pressures just above this phase transition large (several tens of micrometers) bright circular domains (fig. 2b). Interestingly, both of these bright domains disappear on increasing the pressure. Together with the bright domains, above this phase
-51 0.0
I
I
I
0.5
1.0
1.5
AREA /MOLECULE
,
2.0 [nm’l
1
2.5
Fig. 1. Surface pressure-mean molecular area isotherm of DPPENBD at 20°C. On the insert is shown the small-angle X-ray diffraction for a LB film consisting of 21 monolayers deposited at a pressure of SO mN/m.
324
LETTERS
5 June 1992
transition small dark crystallization centers appear (fig. 2b). On increasing the surface pressure, these centers grow up like circular higher-order domains (fig. 2~). On further increase of the pressure these domains grow like dendrites and a second population of crystallization centers appear (fig. 2d). At the next phase transition around 26 mN/m the higherorder domains overcome the energetic barrier and start to merge (fig. 2e). This is the first time such a coexistence of two different types of domains as on fig. 2b has been observed. The bright domains can be interpreted either as several monolayers piling up over another or as domains in which the NBD group is projecting out of the water, thus greatly increasing its fluorescence. The second proposal is more probable because the conformation with the NBD group projecting out of the water is with a larger area per molecule and consequently on increase of the surface pressure it should disappear. Qualitatively, the textures in figs. 2b, 2c and 2d can be understood as an interplay of growth kinetics, line energy of the fluid/ordered interface and longrange electrostatic repulsion [ 7 1. For example the change from circular to a dendritic shape of the higher-order domains (fig. 2d) is due to the increased long-range repulsive electrostatic forces within the domain on increase of its area, as the kinetic factors should not be taken into account because of the slow equilibrium compression. The only possible mechanism of contrast in these observations can be the self-quenching of fluorescence when molecules are closely packed. This statement is supported by the observation that fluorescence of the LB films from DPPE-NBD has much lower intensity than in chloroform solution (not shown). There is evidence in the literature that for small unilamellar vesicles the clustering at the phase transition liquid-expanded-liquid-condensed reduces the fluorescence intensity more than six times due to self-quenching [ 8 1. It seems that quenching of the fluorescence on interaction of the NBD group with water [2] simply regularly decreases the total fluorescence of this compound as the NBD group is in permanent contact with water with the one possible exception mentioned above. Further information on the dark domains’ structure can be gained from LB films deposited at pres-
Volume 193, number 5
CHEMICAL PHYSICS LETTERS
5 June 1992
Fig. 2. Domain structure of DPPE-NBD at a temperature of 20°C and different surface pressures: (a) liquid-gas coexistence region P=O.2 mN/m, A ~2.2 nmz/mofecuie; (b) coexistence of two different types of domains just above the main phase transition P=8.3; (c) circular ordered domains, P~9.8 mN/m; (d) coexistence of dendritic domains and a second population of ordered domains, P= 18 mN/m; (e) merging of ordered domains just above the second phase transition, P=27 mN/m.
325
Volume 193. number 5
CHEMICAL PHYSICS LETTERS
sures above 26 mN/m. Details of transfer at different conditions and the quality of the structures achieved are given elsewhere [ 9 1. From the position of the diffraction maximum (the insert in fig. 1) for a sample transferred at 50 mN/m a bilayer spacing is calculated to be 5.60&O. 1 nm. Reasonable assumptions are that the tails are in the all-trans configuration and no interdigitation of tails exists. For a molecule with fully extended tails oriented normal to the substrate the total length is 3.09 nm [ 91. This, compared to the measured value of 2.80 nm, gives a tail tilt of 3 lo and a projected tails area of 0.45 nm*. This is in good agreement with the measured area per molecule of 0.44 nm2 for 50 mN/m (fig. 1) . Both X-ray (the shift of the diffraction maximum) and FTIR spectroscopy (comparison of intensities of ya(CH3) at 2962 cm-’ and K(CH,) at 2918 cm-i) provide evidence that tail tilt decreases and the order of the structures increases on increasing the pressure for deposition. From the fluorescence microscopy data it is seen that on increasing the pressure above 26 mN/m the liquid phase gradually disappears and correspondingly the order should increase.
Acknowledgement
Help with the measuring techniques is appreciated
326
5 June 1992
from W. Frey (TU, Munich), V. Erochin (Institute of Crystallography, Moscow), S. &stein (Institut fIlr Physikalische Chemie, Mainz) and D. Tsankov (Institute of Organic Chemistry, Sofia). Fluorescence microscopy measurements were carried out in the Laboratory of Professor H. Mohwald (Institut fir Physikalische Chemie, Mainz) following his kind invitation. Many helpful discussions with Professor A.G. Petrov (Institute of Solid State Physics, Sofia) are kindly appreciated. This work was supported under contracts with the National Science Foundation MU-NM-12 and F19.
References [ I] H. Mohwald, Thin Solid Films 159 ( 1988) 1. [2] H. Bercegol, F. Gallet, D. Langevin and J. Meunier, J. Phys. (Paris) 50 (1989) 2277. [ 3 ] P. Muller and F. Gallet, Phys. Rev. Letters 67 ( I99 I ) 1106. [4] G.R. Ivanov, A.T. Todorov and A.G. Petrov, Makromol. Chem. Macromol. Symp. 46 ( 1991) 377. [ 51 G.R. Ivanov, KG. Kostadinov and A.G. Petrov, Thin Solid Films 207 ( 1992), in press. [6] G.R. lvanov and A.G. Petrov, Mol. Ctyst. Liquid Cryst., in press. [7] H. Mohwald, S. Kirstein, H. Haas and M. Fliirsheimer, J. Chim. Phys. 85 (1988) 1009. [8] J.R. Wiener, Biochemistry 24 (1985) 7651. [ 91 G.R. Ivanov and J. Petkova, submitted for publication.