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Imaging and spectroscopy of terrylene molecules isolated in vapor-deposited n-alkane matrices
Chemical Physics 247 Ž1999. 35–40 www.elsevier.nlrlocaterchemphys
Imaging and spectroscopy of terrylene molecules isolated in vapor-deposited n-alkane matrices Jerzy Sepioł 1, Alexander Starukhin 2 , Tatiana Yu. Latychevskaia ) , Jan Jasny 1, Alois Renn, Urs P. Wild Physical Chemistry Laboratory, Swiss Federal Institute of Technology ETH-Zentrum, CH-8092 Zurich, Switzerland Received 22 December 1998
Abstract The matrix isolation technique was applied for producing very thin samples of terrylene doped n-alkane solids directly on the surface of a microscope objective. Fluorescence microscopy allowed the study of the spectroscopic properties of many single terrylene molecules in parallel. From images recorded sequentially at different laser excitation frequencies, the spectral line profiles of corresponding zero-phonon absorption lines were measured. For the two matrices n-decane and n-hexane the line width distributions were determined. Under similar excitation conditions, the individual terrylene molecules were ‘burned out’ much faster in n-hexane than in n-decane matrices. q 1999 Elsevier Science B.V. All rights reserved.
1. Introduction Single molecule spectroscopy and detection have matured to extremely powerful instruments in basic investigations of molecular properties and guest–host interaction on a microscopic scale w1x. Many molecular properties such as low temperature optical line widths have been shown to be distributed. As a consequence, classical bulk measurements determine rather average values, whereas single molecule spectroscopy allows the measurement of individual
)
Corresponding author. Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44r52, 01-224 Warsaw, Poland. 2 Institute of Molecular and Atomic Physics, National Academy of Sciences, F. Scaryna Avenue 70, 220072 Minsk, Belarus. 1
molecular properties prior to the averaging process inherent in bulk measurements w2x. Fluorescence microscopy of single molecules w3x is especially suited to investigate properties which are heterogeneously distributed and allows the investigation of a whole set of molecules in parallel under identical conditions. Results of such measurements can be directly compared to bulk measurements w4x. Since 1990, when fluorescence detection experiments of single pentacene guest molecules embedded in para-terphenyl crystal were started w5x, a number of compounds in different matrices have been successfully investigated w6x. Among the extensively studied guest–host combinations are those prepared by the Shpol’skii frozen-solution method; e.g. terrylene in n-hexadecane w7–9x, in n-dodecane w10x, in n-decane w9x, in an n-decane–n-dodecane mixture w11x, and in n-octane w12x. Isolation and detection of
0301-0104r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 0 1 0 4 Ž 9 9 . 0 0 0 9 9 - 3
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J. Sepioł et al.r Chemical Physics 247 (1999) 35–40
single molecules is achieved with strongly reduced sample volume and concentration. As a rule, detection and spectroscopy of single molecules was performed on samples thinner than 10 mm and having a concentration of guest molecules lower than 10y6 M. Preparation of suitable samples by the Shpol’skii method, which is easy for long chain n-alkanes Že.g. n-hexadecane, n-dodecane., becomes difficult for shorter chain n-alkanes because of the increasing rate of solvent evaporation w13x. An attractive way to study single molecules in matrices of volatile solvents or gases seems to be an application of the well-known matrix-isolation technique w14,15x. In this paper, we report the first spectroscopic results for terrylene molecules in n-alkane matrices prepared by the matrix-isolation technique. The gaseous mixture was deposited directly on the surface of a mirror objective attached to a cold finger. A video camera allowed the investigation of several single molecules in parallel w10,12x.
2. Experimental Low-temperature matrices doped with terrylene were deposited directly on the objective surface. A mirror objective similar to that described in Ref. w10x, but with increased numerical aperture ŽN.A.s 1., smaller magnification Ž80., and a resolution of about 3 mm was mounted in a holder made of copper and attached to a cold finger inside the optical cryostat Žmanufactured at the Institute of Physics of the Polish Academy of Sciences, Warsaw.. The thermal contact between the objective and the holder was improved by using silicone grease ŽBayer, mittelviskos.. One of the four external quartz windows of the cryostat was replaced by a deposition tube equipped with a heatable glass furnace. The matrix deposition procedure was as follows: the cold finger with the attached objective was precooled by cold helium gas to a temperature of 20–40 K below the melting point of the solvent to be deposited. For the two matrices, n-decane and nhexane, the temperature of the cold finger during the deposition was kept at 210 K and 140 K, respectively. Terrylene was heated inside the furnace to about 470 K and at the same time the flow of the n-alkane vapor Žbeing in equilibrium with the de-
gassed liquid. was started. Vapor pressures of n-decane and n-hexane in the gas handling system Ž0.75 l volume. were about 1 and 200 mbar, respectively. During a start up time of 15 min Žthe time period needed for the furnace to reach working temperature. the matrix was deposited on the side-wall of the copper holder. Then the cold finger was rotated by 908, which allowed the matrix to be deposited on the surface of the objective. After 1 min, the cold finger was rotated back and the presence of terrylene was checked by fluorescence excitation using a tunable dye laser and a video camera for detection. In the case when fluorescence was not clearly observable, the deposition procedure was repeated. The roughly estimated upper limit for the ratio guestrhost molecules was 1:1000. After finishing the deposition, the inside of the cold finger was filled with liquid helium. The helium vapor was pumped off and a temperature of about 1.4 K was reached. The optical set-up was similar to the one described in Ref. w10x. The beam from a single mode dye laser with Rh6G dye Žbandwidth about 2 MHz. was focused on the matrix close to the optical axis of the objective with a spot diameter of 50 mm. Rayleigh-scattered pump radiation was blocked by Schott RG610 glass filters placed between the cryostat window and the camera. Resulting images, produced by individual fluorescing molecules, were recorded by a video camera with an image intensifier ŽHammamatsu C2400-25.. To obtain the spectral positions and line shapes of individual molecular resonances, the laser was scanned over 4 GHz in 4 MHz steps with an integration time of 0.32 s Ž8 video frames. per image at each frequency point. To characterize the sample by its basic spectral properties, such as positions and spectral widths of inhomogeneous bands in the fluorescence excitation spectrum, we used a different experimental set-up. The video camera was replaced by a photomultiplier working in the single photon counting mode and the fluorescence signal proportional to the number of excited molecules was measured as a function of wavelength in the spectral range covered by the laser dye. The laser was tuned manually by turning the Birefringent filter, resulting in discrete spectral steps determined by the free spectral range of the thin intracavity etalon Ž; 0.3 nm.. Despite this being a rather crude method, its accuracy is sufficient enough
J. Sepioł et al.r Chemical Physics 247 (1999) 35–40
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to locate and identify the inhomogeneously broadened zero phonon lines of different sites. Spectra measured this way are hereafter called discrete fluorescence excitation ŽDFE. spectra. Emission and excitation spectra of terrylene in Shpol’skii matrices were measured on a fully computer-controlled total luminescence spectroscopy ŽTLS. system consisting of two double-grating SPEX 1402 monochromators. A detailed description of this experimental set-up was given elsewhere w16x.
3. Results and discussion Of the several available guest–host combinations for which single molecule spectroscopy was investigated w1,6x, the terrylene–n-decane system appeared to be the most promising for starting the experiments in vapor-deposited ŽVD. matrices. Studies of the terrylene fluorescence in bulk frozen n-alkane solutions indicated that n-decane is one of the best matrices for this molecule w16x. Recent studies of such matrices have shown that single terrylene molecules can easily be detected in the rather broad temperature range of 2–8 K w9x. Since the cooling conditions for individual molecules in a VD matrix exposed to the vacuum Žnot immersed in superfluid helium. were difficult to predict, it seemed reasonable to perform first experiments with a combination for which non-critical temperature behavior was expected. Fig. 1 Žspectrum 1. shows the fluorescence excitation spectrum of terrylene in n-decane prepared by the Shpol’skii frozen-solution method and measured with the TLS apparatus w16x. According to the notation introduced earlier, the three bands with maxima at 17 612, 17 436 and 17 363 cmy1 were identified as the 0–0 transitions of terrylene isolated in three different sites A, C and B, respectively. To characterize the sample produced by the co-deposition of terrylene and n-decane vapor on the objective surface we measured the DFE spectrum Žsee Section 2.. The DFE spectrum Žspectrum 2. shows the two bands corresponding to the B and C sites in spectrum 1. The similarity between both spectra was expected in accordance with earlier reports on aromatic hydrocarbons isolated in Shpol’skii matrices and in VD
Fig. 1. Fluorescence excitation spectra of terrylene in: Ža. Shpol’skii n-decane matrix Ž ;8 K., measured by the TLS apparatus at a fluorescence wavelength of 600 nm; Žb. vapor-deposited n-decane matrix; spectrum obtained with the discrete fluorescence excitation ŽDFE. method described in Section 2.
n-alkane matrices w15x. However, both bands of the DFE spectrum are significantly broader than the corresponding features in the Shpol’skii n-decane matrix. This might indicate that our sample preparation procedure did not comply with the specific requirements for effective annealing necessary to produce the ‘Shpol’skii effect’ in vapor-deposited n-alkane matrices as demonstrated by Tokousbalides et al. w17x. However, in many experiments performed with very thin samples, it has been shown that thermal and mechanical strain is directly transferred to the molecular environment, leading to a considerable broadening of inhomogeneous bands w18x. Fig. 2 shows a selected image from a data cube consisting of a thousand fluorescence microscopy images recorded while scanning the single-mode dye laser frequency over the range of 4 GHz with a step width of 4 MHz. The fluorescence intensity is plotted as a function of spatial position. The overall size of the investigated sample area is 200 mm = 200 mm. The laser frequency was in resonance with one molecule Ždenoted a . which appeared as a strong peak on a relatively low background. Smaller peaks resulted from molecules which are slightly off resonance. The laser beam intensity of at least 0.3 Wrcm2 was necessary to give fluorescence images of reasonable quality. Under such conditions, the detected molecules were rather ‘stable’, i.e., spectral jumps were seldom observed.
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Fig. 2. Fluorescence intensity of a single terrylene molecule in a VD n-decane matrix observed with the camera. The spatially resolved emission coming from a section of matrix at the fixed excitation wavelength of 573.952 nm Žintensity 0.3 Wrcm2 , accumulation time 0.32 s. is assigned to the fluorescence of a single molecule. The inset shows the spectral dependence of fluorescence intensity observed when integrating the signal from molecule a and plotting the result as a function of the laser detuning. The lineshape can be approximated with a Lorentzian function having a width of 50 MHz ŽFWHM..
Assuming an effective saturation intensity of the order of 1 Wrcm2 Žas reported for the system of terrylene in n-hexadecane w8x., one estimates the power broadening of spectral lines to be lower than 15%. Analysis of the fluorescence coming from a single molecule Ži.e. from a given spot on the detector surface. as a function of the laser frequency allows one to get the spectral profiles of the resonant transition. This can be obtained for several molecules in the field of view. Such an analysis, for the molecule a , is presented in the inset of Fig. 2. The video frames were digitized in real time Ž8 bit. and 8 frames were summed up to give a fluorescence image. The count rates obtained by this procedure do not give the number of photons measured, thus the fluorescence intensity is given in arbitrary units. It is well known that the experimentally measured single molecule excitation Žabsorption. lines are zero-phonon-lines with a Lorentzian shape w1x. In fact, the line-shape of the molecule a was well reproduced by a Lorentzian fit with the spectral width ŽFWHM. of 50 MHz. By taking advantage of the parallel detection of many molecules during one experiment, reasonable statistics for the line width distribution could be made.
Fig. 3a presents a histogram for single terrylene molecules isolated in the vapor-deposited n-decane matrix for a scan of 4 GHz at a wavelength of 573.952 nm. The line widths of 35 molecules are distributed in the frequency range 40–100 MHz. The histogram, showing the number of molecules as function of line width with an interval size of 10 MHz, has a characteristic lower cutoff at 40 MHz, which is close to the life-time-limited line-width of terrylene w8x. According to the histogram, the most probable line width of terrylene in VD n-decane matrix is 60 MHz. For comparison, the histogram in the inset of in Fig. 3a shows the line-width distribution of terrylene as measured recently in a Shpol’skii n-decane matrix at a temperature of 2 K w9x. Systematic temperature studies of this system have shown a much slower increase of the average line-width with temperature Ž40 MHz at 2 K, 55 MHz at 4 K, 65 MHz at 5 K, 130 MHz at 6 K, and 450 MHz at 8 K. than for the Shpol’skii system of terrylene in n-hexadecane w19x. The line-width distribution of the VD system appears to be shifted to larger values by 10–20 MHz. This small shift is rather surprising given the non-standard manner of cooling the VD matrix, via contact with
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a few of the molecules were stable enough to allow the recording of evaluable absorption profiles. The instability of single molecules is indicated by the sudden change of fluorescence intensity; such events have been extensively studied for terrylene in Shpol’skii matrices w1,8x. The jumps in fluorescence intensity arise from spectral jumps of the resonance frequency with respect to the exciting laser. The dwell time between jumps very often depends on the excitation rate. To illustrate the temporal behavior of molecules subjected to the continuous laser irradiation Ž0.3 Wrcm2 ., sequential images were recorded. The fate of every single molecule could be analyzed by integrating the fluorescence intensity coming from a small area in the image containing a molecule as a function of time. Fig. 4 presents a typical temporal dependence of fluorescence for two molecules, a and b, from a set of 16 molecules which were originally in resonance with the excitation frequency. The fluorescence intensity of molecule a jumped to zero already after 5 s of irradiation. The fluorescence intensity for molecule b showed strong fluctuations and vanished after 15 s. The averaged intensity for all 16 molecules, denoted Žave., indicates that practically all molecules are ‘burned’ out of resonance after 15 s of excitation. Fig. 3. Ža. Distribution of linewidths for terrylene molecules in a VD n-decane matrix excited by the laser beam with wavelength around 573.952 nm and power of 0.3 Wrcm2 . In the inset, a similar distribution for terrylene molecules in a Shpol’skii n-decane matrix at 2 K w6x is shown for comparison; Žb. histogram of linewidths for terrylene molecules in a VD n-hexane matrix excited at 571.252 nm with a laser beam intensity of 0.3 Wrcm2 .
the quartz objective surface Žnot with the liquid helium.. Based on the experience with Shpol’skii n-decane matrixes at different temperatures, we can conclude that the temperature of our sample was lower than 4 K. We extended our studies to the other much more volatile solvent, n-hexane. A distribution of line widths is shown in Fig. 3b. The histogram is plotted with 20 MHz steps because the spectral stability of the single molecule absorption lines in the VD matrix n-hexane is much lower than that observed in the n-decane matrix, resulting in poor statistics. Only
Fig. 4. The temporal behavior of the fluorescence for terrylene molecules in a vapor-deposited n-hexane matrix under continuous irradiation at 571.247 nm with a laser beam intensity of 0.3 Wrcm2 . The individual emission of two molecules Ža and b. and the average emission for a set of 16 molecules, denoted average Ž16., are presented.
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The irradiation of terrylene molecules in the VD n-decane matrix showed stable molecules which were observed for a longer time than minutes. When the laser intensity was increased by a factor of 30, the average fluorescence intensity decreased by a factor of two within 20 s. In spite of large differences in stability, the line-width distribution of terrylene in VD n-hexane matrix was similar to that observed in the VD n-decane. The most probable line width is also in the range 50–70 MHz, suggesting that fast spectral diffusion is still much less effective than that observed for terrylene in polymeric materials where the line widths were at least one order of magnitude broader w13x. In conclusion, the matrix isolation technique was successfully applied to the spectroscopic studies of single terrylene molecules in n-alkanes. Both the concentration of guest molecules and the thickness of a matrix deposited directly on the objective surface can be easily controlled. The technique opens up the possibility of using new materials, including volatile solvents and Žrare. gases, as matrices for single-molecule spectroscopy. Studying the same probe molecules in a variety of host materials should result in a better understanding of ‘nano-environments’ in matrices. Also, the technique of vapor deposition offers the interesting possibility of covering a given matrix with another matrix layer, which could enable studies of energy transfer between individual molecules embedded in different materials.
Acknowledgements We thank Bruno Lambillotte for the construction of the objective holder. We also wish to thank Dr. Robert Kolos for valuable advice concerning the construction of the gas handling system and for
discussions during the preparation of the manuscript. This work was supported by the Swiss National Science Foundation ŽSNF. and ETH Zurich. References w1x Th. Basche, W.E. Moerner, M. Orrit, U.P. Wild ŽEds.., Single-Molecule Optical Detection, Imaging and Spectroscopy, VCH, Weinheim, 1996. w2x W.E. Moerner, Acc. Chem. Res. 29 Ž1996. 563. w3x F. Guttler, T. Irngartinger, T. Plakhotnik, A. Renn, U.P. ¨ Wild, Chem. Phys. Lett. 217 Ž1994. 393. w4x H. Bach, T. Irngartinger, A. Renn, U.P. Wild, Mol. Cryst. Liq. Cryst. 291 Ž1996. 89. w5x M. Orrit, J. Bernard, Phys. Rev. Lett. 65 Ž1990. 2716. w6x T. Plakhotnik, E.A. Donley, U.P. Wild, Annu. Rev. Phys. Chem. 48 Ž1997. 181. w7x T. Plakhotnik, W.E. Moerner, T. Irngartinger, U.P. Wild, Chimia 48 Ž1994. 31. w8x W.E. Moerner, T. Plakhotnik, T. Irngartinger, M. Croci, V. Palm, U.P. Wild, J. Phys. Chem. 98 Ž1994. 7382. w9x T. Irngartinger, A. Renn, G. Zumofen, U.P. Wild, J. Luminesc. 76 Ž1998. 279. w10x J. Jasny, J. Sepiol, T. Irngartinger, M. Traber, A. Renn, U.P. Wild, Rev. Sci. Instrum. 67 Ž1996. 1425. w11x N. Caspary, V. Palm, K. Rebane, V. Bondybey, Chem. Phys. Lett. 283 Ž1998. 345. w12x H. Bach, A. Renn, U.P. Wild, Chem. Phys. Lett. 266 Ž1997. 317. w13x B. Kozankiewicz, J. Bernard, M. Orrit, J. Chem. Phys. 101 Ž1994. 9177. w14x L. Andrews, M. Moskovits ŽEds.., Chemistry and Physics of Matrix-Isolated Species, Elsevier, Amsterdam, 1989. w15x J.R. Maple, E.L. Wehry, G. Mamantov, Anal. Chem. 52 Ž1980. 920. w16x K. Palewska, J. Lipinski, J. Sworakowski, J. Sepiol, H. Gygax, E. Meister, U.P. Wild, J. Phys. Chem. 99 Ž1995. 16835. w17x P. Tokousbalides, E.L. Wehry, G. Mamantov, J. Phys. Chem. 81 Ž1977. 1769. w18x Th. Basche, Chem. Phys. Lett. 225 ´ S. Kummer, C. Brauchle, ¨ Ž1994. 116. w19x T. Irngartinger, Abbildene Einzelmolekul-Spektroskopie von ¨ Terrylen, PhD thesis, ETH-Zurich, Nr. 12305, Zurich, 1997.
Imaging and spectroscopy of terrylene molecules isolated in vapor-deposited n-alkane matrices Jerzy Sepioł 1, Alexander Starukhin 2 , Tatiana Yu. Latychevskaia ) , Jan Jasny 1, Alois Renn, Urs P. Wild Physical Chemistry Laboratory, Swiss Federal Institute of Technology ETH-Zentrum, CH-8092 Zurich, Switzerland Received 22 December 1998
Abstract The matrix isolation technique was applied for producing very thin samples of terrylene doped n-alkane solids directly on the surface of a microscope objective. Fluorescence microscopy allowed the study of the spectroscopic properties of many single terrylene molecules in parallel. From images recorded sequentially at different laser excitation frequencies, the spectral line profiles of corresponding zero-phonon absorption lines were measured. For the two matrices n-decane and n-hexane the line width distributions were determined. Under similar excitation conditions, the individual terrylene molecules were ‘burned out’ much faster in n-hexane than in n-decane matrices. q 1999 Elsevier Science B.V. All rights reserved.
1. Introduction Single molecule spectroscopy and detection have matured to extremely powerful instruments in basic investigations of molecular properties and guest–host interaction on a microscopic scale w1x. Many molecular properties such as low temperature optical line widths have been shown to be distributed. As a consequence, classical bulk measurements determine rather average values, whereas single molecule spectroscopy allows the measurement of individual
)
Corresponding author. Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44r52, 01-224 Warsaw, Poland. 2 Institute of Molecular and Atomic Physics, National Academy of Sciences, F. Scaryna Avenue 70, 220072 Minsk, Belarus. 1
molecular properties prior to the averaging process inherent in bulk measurements w2x. Fluorescence microscopy of single molecules w3x is especially suited to investigate properties which are heterogeneously distributed and allows the investigation of a whole set of molecules in parallel under identical conditions. Results of such measurements can be directly compared to bulk measurements w4x. Since 1990, when fluorescence detection experiments of single pentacene guest molecules embedded in para-terphenyl crystal were started w5x, a number of compounds in different matrices have been successfully investigated w6x. Among the extensively studied guest–host combinations are those prepared by the Shpol’skii frozen-solution method; e.g. terrylene in n-hexadecane w7–9x, in n-dodecane w10x, in n-decane w9x, in an n-decane–n-dodecane mixture w11x, and in n-octane w12x. Isolation and detection of
0301-0104r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 1 - 0 1 0 4 Ž 9 9 . 0 0 0 9 9 - 3
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J. Sepioł et al.r Chemical Physics 247 (1999) 35–40
single molecules is achieved with strongly reduced sample volume and concentration. As a rule, detection and spectroscopy of single molecules was performed on samples thinner than 10 mm and having a concentration of guest molecules lower than 10y6 M. Preparation of suitable samples by the Shpol’skii method, which is easy for long chain n-alkanes Že.g. n-hexadecane, n-dodecane., becomes difficult for shorter chain n-alkanes because of the increasing rate of solvent evaporation w13x. An attractive way to study single molecules in matrices of volatile solvents or gases seems to be an application of the well-known matrix-isolation technique w14,15x. In this paper, we report the first spectroscopic results for terrylene molecules in n-alkane matrices prepared by the matrix-isolation technique. The gaseous mixture was deposited directly on the surface of a mirror objective attached to a cold finger. A video camera allowed the investigation of several single molecules in parallel w10,12x.
2. Experimental Low-temperature matrices doped with terrylene were deposited directly on the objective surface. A mirror objective similar to that described in Ref. w10x, but with increased numerical aperture ŽN.A.s 1., smaller magnification Ž80., and a resolution of about 3 mm was mounted in a holder made of copper and attached to a cold finger inside the optical cryostat Žmanufactured at the Institute of Physics of the Polish Academy of Sciences, Warsaw.. The thermal contact between the objective and the holder was improved by using silicone grease ŽBayer, mittelviskos.. One of the four external quartz windows of the cryostat was replaced by a deposition tube equipped with a heatable glass furnace. The matrix deposition procedure was as follows: the cold finger with the attached objective was precooled by cold helium gas to a temperature of 20–40 K below the melting point of the solvent to be deposited. For the two matrices, n-decane and nhexane, the temperature of the cold finger during the deposition was kept at 210 K and 140 K, respectively. Terrylene was heated inside the furnace to about 470 K and at the same time the flow of the n-alkane vapor Žbeing in equilibrium with the de-
gassed liquid. was started. Vapor pressures of n-decane and n-hexane in the gas handling system Ž0.75 l volume. were about 1 and 200 mbar, respectively. During a start up time of 15 min Žthe time period needed for the furnace to reach working temperature. the matrix was deposited on the side-wall of the copper holder. Then the cold finger was rotated by 908, which allowed the matrix to be deposited on the surface of the objective. After 1 min, the cold finger was rotated back and the presence of terrylene was checked by fluorescence excitation using a tunable dye laser and a video camera for detection. In the case when fluorescence was not clearly observable, the deposition procedure was repeated. The roughly estimated upper limit for the ratio guestrhost molecules was 1:1000. After finishing the deposition, the inside of the cold finger was filled with liquid helium. The helium vapor was pumped off and a temperature of about 1.4 K was reached. The optical set-up was similar to the one described in Ref. w10x. The beam from a single mode dye laser with Rh6G dye Žbandwidth about 2 MHz. was focused on the matrix close to the optical axis of the objective with a spot diameter of 50 mm. Rayleigh-scattered pump radiation was blocked by Schott RG610 glass filters placed between the cryostat window and the camera. Resulting images, produced by individual fluorescing molecules, were recorded by a video camera with an image intensifier ŽHammamatsu C2400-25.. To obtain the spectral positions and line shapes of individual molecular resonances, the laser was scanned over 4 GHz in 4 MHz steps with an integration time of 0.32 s Ž8 video frames. per image at each frequency point. To characterize the sample by its basic spectral properties, such as positions and spectral widths of inhomogeneous bands in the fluorescence excitation spectrum, we used a different experimental set-up. The video camera was replaced by a photomultiplier working in the single photon counting mode and the fluorescence signal proportional to the number of excited molecules was measured as a function of wavelength in the spectral range covered by the laser dye. The laser was tuned manually by turning the Birefringent filter, resulting in discrete spectral steps determined by the free spectral range of the thin intracavity etalon Ž; 0.3 nm.. Despite this being a rather crude method, its accuracy is sufficient enough
J. Sepioł et al.r Chemical Physics 247 (1999) 35–40
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to locate and identify the inhomogeneously broadened zero phonon lines of different sites. Spectra measured this way are hereafter called discrete fluorescence excitation ŽDFE. spectra. Emission and excitation spectra of terrylene in Shpol’skii matrices were measured on a fully computer-controlled total luminescence spectroscopy ŽTLS. system consisting of two double-grating SPEX 1402 monochromators. A detailed description of this experimental set-up was given elsewhere w16x.
3. Results and discussion Of the several available guest–host combinations for which single molecule spectroscopy was investigated w1,6x, the terrylene–n-decane system appeared to be the most promising for starting the experiments in vapor-deposited ŽVD. matrices. Studies of the terrylene fluorescence in bulk frozen n-alkane solutions indicated that n-decane is one of the best matrices for this molecule w16x. Recent studies of such matrices have shown that single terrylene molecules can easily be detected in the rather broad temperature range of 2–8 K w9x. Since the cooling conditions for individual molecules in a VD matrix exposed to the vacuum Žnot immersed in superfluid helium. were difficult to predict, it seemed reasonable to perform first experiments with a combination for which non-critical temperature behavior was expected. Fig. 1 Žspectrum 1. shows the fluorescence excitation spectrum of terrylene in n-decane prepared by the Shpol’skii frozen-solution method and measured with the TLS apparatus w16x. According to the notation introduced earlier, the three bands with maxima at 17 612, 17 436 and 17 363 cmy1 were identified as the 0–0 transitions of terrylene isolated in three different sites A, C and B, respectively. To characterize the sample produced by the co-deposition of terrylene and n-decane vapor on the objective surface we measured the DFE spectrum Žsee Section 2.. The DFE spectrum Žspectrum 2. shows the two bands corresponding to the B and C sites in spectrum 1. The similarity between both spectra was expected in accordance with earlier reports on aromatic hydrocarbons isolated in Shpol’skii matrices and in VD
Fig. 1. Fluorescence excitation spectra of terrylene in: Ža. Shpol’skii n-decane matrix Ž ;8 K., measured by the TLS apparatus at a fluorescence wavelength of 600 nm; Žb. vapor-deposited n-decane matrix; spectrum obtained with the discrete fluorescence excitation ŽDFE. method described in Section 2.
n-alkane matrices w15x. However, both bands of the DFE spectrum are significantly broader than the corresponding features in the Shpol’skii n-decane matrix. This might indicate that our sample preparation procedure did not comply with the specific requirements for effective annealing necessary to produce the ‘Shpol’skii effect’ in vapor-deposited n-alkane matrices as demonstrated by Tokousbalides et al. w17x. However, in many experiments performed with very thin samples, it has been shown that thermal and mechanical strain is directly transferred to the molecular environment, leading to a considerable broadening of inhomogeneous bands w18x. Fig. 2 shows a selected image from a data cube consisting of a thousand fluorescence microscopy images recorded while scanning the single-mode dye laser frequency over the range of 4 GHz with a step width of 4 MHz. The fluorescence intensity is plotted as a function of spatial position. The overall size of the investigated sample area is 200 mm = 200 mm. The laser frequency was in resonance with one molecule Ždenoted a . which appeared as a strong peak on a relatively low background. Smaller peaks resulted from molecules which are slightly off resonance. The laser beam intensity of at least 0.3 Wrcm2 was necessary to give fluorescence images of reasonable quality. Under such conditions, the detected molecules were rather ‘stable’, i.e., spectral jumps were seldom observed.
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Fig. 2. Fluorescence intensity of a single terrylene molecule in a VD n-decane matrix observed with the camera. The spatially resolved emission coming from a section of matrix at the fixed excitation wavelength of 573.952 nm Žintensity 0.3 Wrcm2 , accumulation time 0.32 s. is assigned to the fluorescence of a single molecule. The inset shows the spectral dependence of fluorescence intensity observed when integrating the signal from molecule a and plotting the result as a function of the laser detuning. The lineshape can be approximated with a Lorentzian function having a width of 50 MHz ŽFWHM..
Assuming an effective saturation intensity of the order of 1 Wrcm2 Žas reported for the system of terrylene in n-hexadecane w8x., one estimates the power broadening of spectral lines to be lower than 15%. Analysis of the fluorescence coming from a single molecule Ži.e. from a given spot on the detector surface. as a function of the laser frequency allows one to get the spectral profiles of the resonant transition. This can be obtained for several molecules in the field of view. Such an analysis, for the molecule a , is presented in the inset of Fig. 2. The video frames were digitized in real time Ž8 bit. and 8 frames were summed up to give a fluorescence image. The count rates obtained by this procedure do not give the number of photons measured, thus the fluorescence intensity is given in arbitrary units. It is well known that the experimentally measured single molecule excitation Žabsorption. lines are zero-phonon-lines with a Lorentzian shape w1x. In fact, the line-shape of the molecule a was well reproduced by a Lorentzian fit with the spectral width ŽFWHM. of 50 MHz. By taking advantage of the parallel detection of many molecules during one experiment, reasonable statistics for the line width distribution could be made.
Fig. 3a presents a histogram for single terrylene molecules isolated in the vapor-deposited n-decane matrix for a scan of 4 GHz at a wavelength of 573.952 nm. The line widths of 35 molecules are distributed in the frequency range 40–100 MHz. The histogram, showing the number of molecules as function of line width with an interval size of 10 MHz, has a characteristic lower cutoff at 40 MHz, which is close to the life-time-limited line-width of terrylene w8x. According to the histogram, the most probable line width of terrylene in VD n-decane matrix is 60 MHz. For comparison, the histogram in the inset of in Fig. 3a shows the line-width distribution of terrylene as measured recently in a Shpol’skii n-decane matrix at a temperature of 2 K w9x. Systematic temperature studies of this system have shown a much slower increase of the average line-width with temperature Ž40 MHz at 2 K, 55 MHz at 4 K, 65 MHz at 5 K, 130 MHz at 6 K, and 450 MHz at 8 K. than for the Shpol’skii system of terrylene in n-hexadecane w19x. The line-width distribution of the VD system appears to be shifted to larger values by 10–20 MHz. This small shift is rather surprising given the non-standard manner of cooling the VD matrix, via contact with
J. Sepioł et al.r Chemical Physics 247 (1999) 35–40
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a few of the molecules were stable enough to allow the recording of evaluable absorption profiles. The instability of single molecules is indicated by the sudden change of fluorescence intensity; such events have been extensively studied for terrylene in Shpol’skii matrices w1,8x. The jumps in fluorescence intensity arise from spectral jumps of the resonance frequency with respect to the exciting laser. The dwell time between jumps very often depends on the excitation rate. To illustrate the temporal behavior of molecules subjected to the continuous laser irradiation Ž0.3 Wrcm2 ., sequential images were recorded. The fate of every single molecule could be analyzed by integrating the fluorescence intensity coming from a small area in the image containing a molecule as a function of time. Fig. 4 presents a typical temporal dependence of fluorescence for two molecules, a and b, from a set of 16 molecules which were originally in resonance with the excitation frequency. The fluorescence intensity of molecule a jumped to zero already after 5 s of irradiation. The fluorescence intensity for molecule b showed strong fluctuations and vanished after 15 s. The averaged intensity for all 16 molecules, denoted Žave., indicates that practically all molecules are ‘burned’ out of resonance after 15 s of excitation. Fig. 3. Ža. Distribution of linewidths for terrylene molecules in a VD n-decane matrix excited by the laser beam with wavelength around 573.952 nm and power of 0.3 Wrcm2 . In the inset, a similar distribution for terrylene molecules in a Shpol’skii n-decane matrix at 2 K w6x is shown for comparison; Žb. histogram of linewidths for terrylene molecules in a VD n-hexane matrix excited at 571.252 nm with a laser beam intensity of 0.3 Wrcm2 .
the quartz objective surface Žnot with the liquid helium.. Based on the experience with Shpol’skii n-decane matrixes at different temperatures, we can conclude that the temperature of our sample was lower than 4 K. We extended our studies to the other much more volatile solvent, n-hexane. A distribution of line widths is shown in Fig. 3b. The histogram is plotted with 20 MHz steps because the spectral stability of the single molecule absorption lines in the VD matrix n-hexane is much lower than that observed in the n-decane matrix, resulting in poor statistics. Only
Fig. 4. The temporal behavior of the fluorescence for terrylene molecules in a vapor-deposited n-hexane matrix under continuous irradiation at 571.247 nm with a laser beam intensity of 0.3 Wrcm2 . The individual emission of two molecules Ža and b. and the average emission for a set of 16 molecules, denoted average Ž16., are presented.
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The irradiation of terrylene molecules in the VD n-decane matrix showed stable molecules which were observed for a longer time than minutes. When the laser intensity was increased by a factor of 30, the average fluorescence intensity decreased by a factor of two within 20 s. In spite of large differences in stability, the line-width distribution of terrylene in VD n-hexane matrix was similar to that observed in the VD n-decane. The most probable line width is also in the range 50–70 MHz, suggesting that fast spectral diffusion is still much less effective than that observed for terrylene in polymeric materials where the line widths were at least one order of magnitude broader w13x. In conclusion, the matrix isolation technique was successfully applied to the spectroscopic studies of single terrylene molecules in n-alkanes. Both the concentration of guest molecules and the thickness of a matrix deposited directly on the objective surface can be easily controlled. The technique opens up the possibility of using new materials, including volatile solvents and Žrare. gases, as matrices for single-molecule spectroscopy. Studying the same probe molecules in a variety of host materials should result in a better understanding of ‘nano-environments’ in matrices. Also, the technique of vapor deposition offers the interesting possibility of covering a given matrix with another matrix layer, which could enable studies of energy transfer between individual molecules embedded in different materials.
Acknowledgements We thank Bruno Lambillotte for the construction of the objective holder. We also wish to thank Dr. Robert Kolos for valuable advice concerning the construction of the gas handling system and for
discussions during the preparation of the manuscript. This work was supported by the Swiss National Science Foundation ŽSNF. and ETH Zurich. References w1x Th. Basche, W.E. Moerner, M. Orrit, U.P. Wild ŽEds.., Single-Molecule Optical Detection, Imaging and Spectroscopy, VCH, Weinheim, 1996. w2x W.E. Moerner, Acc. Chem. Res. 29 Ž1996. 563. w3x F. Guttler, T. Irngartinger, T. Plakhotnik, A. Renn, U.P. ¨ Wild, Chem. Phys. Lett. 217 Ž1994. 393. w4x H. Bach, T. Irngartinger, A. Renn, U.P. Wild, Mol. Cryst. Liq. Cryst. 291 Ž1996. 89. w5x M. Orrit, J. Bernard, Phys. Rev. Lett. 65 Ž1990. 2716. w6x T. Plakhotnik, E.A. Donley, U.P. Wild, Annu. Rev. Phys. Chem. 48 Ž1997. 181. w7x T. Plakhotnik, W.E. Moerner, T. Irngartinger, U.P. Wild, Chimia 48 Ž1994. 31. w8x W.E. Moerner, T. Plakhotnik, T. Irngartinger, M. Croci, V. Palm, U.P. Wild, J. Phys. Chem. 98 Ž1994. 7382. w9x T. Irngartinger, A. Renn, G. Zumofen, U.P. Wild, J. Luminesc. 76 Ž1998. 279. w10x J. Jasny, J. Sepiol, T. Irngartinger, M. Traber, A. Renn, U.P. Wild, Rev. Sci. Instrum. 67 Ž1996. 1425. w11x N. Caspary, V. Palm, K. Rebane, V. Bondybey, Chem. Phys. Lett. 283 Ž1998. 345. w12x H. Bach, A. Renn, U.P. Wild, Chem. Phys. Lett. 266 Ž1997. 317. w13x B. Kozankiewicz, J. Bernard, M. Orrit, J. Chem. Phys. 101 Ž1994. 9177. w14x L. Andrews, M. Moskovits ŽEds.., Chemistry and Physics of Matrix-Isolated Species, Elsevier, Amsterdam, 1989. w15x J.R. Maple, E.L. Wehry, G. Mamantov, Anal. Chem. 52 Ž1980. 920. w16x K. Palewska, J. Lipinski, J. Sworakowski, J. Sepiol, H. Gygax, E. Meister, U.P. Wild, J. Phys. Chem. 99 Ž1995. 16835. w17x P. Tokousbalides, E.L. Wehry, G. Mamantov, J. Phys. Chem. 81 Ž1977. 1769. w18x Th. Basche, Chem. Phys. Lett. 225 ´ S. Kummer, C. Brauchle, ¨ Ž1994. 116. w19x T. Irngartinger, Abbildene Einzelmolekul-Spektroskopie von ¨ Terrylen, PhD thesis, ETH-Zurich, Nr. 12305, Zurich, 1997.