Anal. Chem. 1998, 70, 5198-5208
Grouping of Independent Single Molecules on Silicon Surfaces Mitsuru Ishikawa,*,† Osamu Yogi,† Jing Yong Ye,† and Tetsuji Yasuda‡
Joint Research Center for Atom Technology (JRCAT)sAngstrom Technology Partnership (ATP) and JRCATsNational Institute for Advanced Interdisciplinary Research (NAIR), 1-1-4 Higashi, Tsukuba, Ibaraki 305-0046, Japan Yoshihiro Maruyama
Tsukuba Research Laboratory, Hamamatsu Photonics K. K., 5-9-2 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
We evaluated the possibilities of preparing single analyte molecules on silicon surfaces for chemical analysis based on fluorescence measurement. A single-molecule imaging technique involving far-field optical microscopy was used to visualize rhodamine B (rhB) molecules in submonolayers prepared on Si wafers. This technique revealed that fluorescence of solvent-free, adsorbed rhB molecules in submonolayers appeared in two different forms: isolated fluorescent spots in which a countable number of independent single rhB molecules were grouped together and diffused fluorescence in the region with no fluorescent spots. On the basis of the fluorescence imaging and timeresolved fluorometry of individual fluorescent spots, together with examination of light-scattering sites on Si wafers, we discussed the possibility that the isolated fluorescent spots occurred on crystal-originated sites dotted on Si wafers. Our findings should provide guiding principles for developing vials for single analyte molecules on Si surfaces. Recent advances in the imaging and spectroscopy of single molecules based on fluorescence measurement1-15 have attracted wide attention in the physical and biological sciences. At air* Corresponding author: (e-mail)
[email protected]. † JRCAT-ATP. ‡ JRCAT-NAIR. (1) Betzig, E.; Chichester, R. J. Science 1993, 262, 1422-1425. (2) Ambrose, W. P.; Goodwin, P. M.; Martin, J. C.; Keller, R. A. Phys. Rev. Lett. 1994, 72, 160-163. (3) Ishikawa, M.; Hirano, K.; Hayakawa, T.; Hosoi, S.; Brenner, S. Jpn. J. Appl. Phys. 1994, 33, 1571-1576. (4) Xie, X. S.; Dunn, R. C. Science 1994, 265, 361-364. (5) Ambrose, W. P.; Goodwin, P. M.; Martin, J. C.; Keller, R. A. Science 1994, 265, 364-367. (6) Trautman, J. K.; Macklin, J. J.; Brus, L. E.; Betzig, E. Nature 1994, 269, 40-42. (7) Macklin, J. J.; Trautman, J. K.; Harris, T. D.; Brus, L. E. Science 1996, 272, 255-258. (8) Ha, T.; Enderle, Th.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6264-6268. (9) Ha, T.; Enderle, Th.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Phys. Rev. Lett. 1996, 77, 3979-3982. (10) Bopp, M. A.; Meixner, A. J.; Tarrach, G.; Zschokke-Granacher, I.; Novotny, L. Chem. Phys. Lett. 1996, 263, 721-726. (11) Lu, H. P.; Xie, X. S. Nature 1997, 385, 143-146. (12) Lu, H. P.; Xie, X. S. J. Phys. Chem. B 1997, 101, 2753-2757.
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substrate interfaces at room temperature, it is now possible to distinguish dipole orientations1,7,9,10,13,15 and to observe photochemical reactions,2,12 positions,3 excited-state dynamics,4,5,7,14 resonance energy transfer,8 and time-dependent spectral fluctuations6,11 of individual dye molecules. As a future plan to put such research into practical use, we should consider doing singlemolecule chemical analysis particularly at air-substrate interfaces, where single analyte molecules are prepared after being transported. Advantages in using solvent-free surfaces include immobilizing target molecules without Brownian motion in solution and reducing possible photobleaching or fluorescence quenching of target molecules, as can be induced by solvents or dissolved material in the solvents used. In this study, we therefore evaluated fluorescence characteristics of dye molecules in submonolayers on solvent-free substrate surfaces using videomicroscopy plus time-resolved spectrofluorometry. We selected rhodamine B (rhB) submonolayers on mirrorlike Si wafers covered with native oxide instead of on glass or fused quartz for the following reasons. First, rhB is a dye molecule extensively studied not only in solution16-19 but also in adsorption.20-30 Such research, therefore, provides us with many guidelines for using rhB to explore photophysical and photo(13) Ruiter, A. G. T.; Veerman, J. A.; Garcia-Parajo, M. F.; van Hulst, N. F. J. Phys. Chem. A 1997, 101, 7318-7323. (14) Ye, J. Y.; Ishikawa, M.; Yogi, O.; Okada, T.; Maruyama, Y. Chem. Phys. Lett. 1998, 288, 885-890. (15) Bopp, M. A.; Jia, Y.; Haran, G.; Morlino, E. A.; Hochstrasser, R. M. Appl. Phys. Lett. 1998, 73, 6-9. (16) Fo ¨rster, Th.; Ko¨nig, E. Z. Z. Elektrochem. 1957, 61, 344-348. (17) Arbeloa, I. L.; Rohatgi-Mukherjee, K. K. Chem. Phys. Lett. 1986, 129, 607614. (18) Casey, K. G.; Quitevis, E. L. J. Phys. Chem. 1988, 92, 6590-6594. (19) Chang, T.-L.; Cheung, H. C. J. Phys. Chem. 1992, 96, 4874-4878. (20) Nakashima, N.; Yoshihara, K.; Willig, F. J. Chem. Phys. 1980, 73, 35533559. (21) Heinz, T. F.; Chen, C. K.; Ricard, D.; Shen Y. R. Phys. Rev. Lett. 1982, 48, 478-481. (22) Itoh, K.; Chiyokawa, Y.; Nakao, M.; Honda, K. J. Am. Chem. Soc. 1984, 106, 1620-1627. (23) Liang, Y.; Moy, P. F.; Poole, J. A.; Goncalves, A. M. P. J. Phys. Chem. 1984, 88, 2451-2455. (24) Kemnitz, K.; Tamai, N.; Yamazaki, I.; Nakashima, N.; Yoshihara, K. J. Phys. Chem. 1986, 90, 5094-5101. (25) Kemnitz, K.; Tamai, N.; Yamazaki, I.; Nakashima, N.; Yoshihara, K. J. Phys. Chem. 1987, 91, 1423-1430. (26) Kemnitz, K.; Nakashima, N.; Yoshihara, K. J. Phys. Chem. 1988, 92, 39153925. 10.1021/ac980622r CCC: $15.00
© 1998 American Chemical Society Published on Web 11/07/1998
Figure 1. Experimental setup for single-molecule fluorescence imaging experiments. The inset shows the molecular structure and size of rhB.
chemical phenomena in the single-molecule regime. Second, we chose Si wafers because they are often used as substrates for miniaturized total analysis systems (µ-TAS), where structures in the micrometer regime are defined chemically or photolithographically.31-34 Thus, future applications are abundant for Si wafers as substrates on which single analyte molecules are prepared. Indeed, single-molecule chemical analysis is an ultimate goal in the development of µ-TAS technologies. We found that bright fluorescent spots dotted on Si wafers were free from strong quenching and had grouped a countable number of independent single rhB molecules. Because identification of the number of molecules is critical in single-molecule chemical analysis, the independent nature of individual rhB molecules within fluorescent spots on Si surfaces is suitable for analytical applications of the single-molecule imaging technique involved in this study. EXPERIMENTAL SECTION In this study we used single-molecule imaging and spectroscopy based on far-field optical microscopy to examine the distribution of rhB molecules on substrate surfaces. Experiments included (i) acquisition of fluorescence and light-scattering images of the substrate surfaces on which target molecules were prepared, (ii) emission spectroscopy, with or without time resolution, of (27) Hashimoto, K.; Hiramoto, M.; Sakata, T. J. Phys. Chem. 1988, 92, 42724274. (28) Peterson, E. S.; Harris, C. B. J. Chem. Phys. 1989, 91, 2683-2688. (29) Sakata, T.; Hashimoto, K.; Hiramoto, M. J. Phys. Chem. 1990, 94, 30403045. (30) Morgenthaler, M. J. E.; Meech, S. R. J. Phys. Chem. 1996, 100, 33233329. (31) Manz, A.; Miyahara, Y.; Miura, J.; Watanabe, Y.; Miyagi, H. Sato, K. Sens. Actuators 1990, B1, 249-255. (32) Liang, Z.; Chiem, N.; Ocvirl, G.; Tang, T.; Fluri, K.; Harrison, D. J. Anal. Chem. 1996, 68, 1040-1046. (33) Raymond, D. E.; Manz, A.; Widmer, H. M. Anal. Chem. 1996, 68, 25152522. (34) Fister, J. C., III; Jacobson, S. C.; Davis, L. M.; Ramsey, J. M. Anal. Chem. 1998, 70, 431-437.
selected fluorescent spots on the surfaces, and (iii) atomic force microscopy (AFM) to evaluate the irregularities of the surfaces. Instrumentation Used for Single-Molecule Imaging. Figure 1 shows a diagram of the experimental setup used for fluorescence imaging of single rhB molecules. Samples were irradiated using a continuous-wave (cw) cavity-dumped dye laser (Spectra-Physics 375B and 344S) that was synchronously pumped with a cw mode-locked argon ion laser (Spectra-Physics 203018). The pulse dye laser, oscillating at 540 nm, was operated at a 4-MHz repetition rate with a 10-ps full width at half-maximum (fwhm). The laser beam was circularly polarized with a BabinetSoleil compensator (BSC) to irradiate uniformly target molecules. The colatitude of the incident direction of the laser light was ∼65° with respect to the z axis (see Figure 1). The light circularly polarized was in the plane perpendicular to the incident direction. This irradiation is, on the basis of the principle of superposition, equivalent to simultaneous irradiation of two circularly polarized beams whose incident directions are along the x and z axes. We used the picosecond-pulse dye laser because the tunability of the dye laser to the absoption maximum of rhB molecules (∼540 nm) was important. The pulse operation was not important for the imaging experiment, but it was necessary for the time-resolved measurement. A frequency-doubled (532 nm) cw Nd:YAG laser (Coherent, DPSS 532) was also used in experiments involving linearly polarized excitation. We used a photon-counting video camera system (Hamamatsu Argus) composed of a photoncounting camera head (C2400-40), an image intensifier (II) controller (M4253), an image processor (C3930), and a computer. The photon-counting camera head was coupled with an epifluorescence microscope (Nikon Optiphot XP) through a relay lens (RL). The instruments depicted in Figure 1 were housed in a clean-air booth (class 1000). All of the experiments were carried out at room temperature (296 K) in air. Preparation of Submonolayers. Before preparing the submonolayers, we selected clean Si wafers. By clean, we mean that the wafers were not contaminated with light-scattering Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
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particles. When contaminated Si wafers were unavoidable, wiping the surface of the wafers with a piece of lens-cleaning paper (Eastman Kodak) soaked with Cl3CCH3 effectively reduced lightscattering contaminants. The rhB submonolayers were prepared by first placing a piece of lens-cleaning paper on the surface of a 2-in.-diameter 〈100〉 -oriented Si wafer covered with a native oxide. Then, a 50-µL drop of a rhB-methanol solution was placed on the paper at one end of the wafer. Last, the wet paper was gently dragged across the face of the wafer, thus leaving the solvent to naturally evaporate. These procedures (drop-and-drag treatment) using high-quality methanol or acetone instead of dye solutions are also used as cleaning methods for optics such as dielectric mirrors. The submonolayers were prepared using selected concentrations of rhB-methanol solutions: 1.5 × 10-7, 10-6, 10-5, and 10-4 M. Similar preparation procedures were reported in previous studies20,22-26,28,30 involving submonolayers and monolayers of dye molecules. Peterson and Harris28 pointed out that more uniform coverage is obtained with this method than with the spin-coating method21 when low-viscosity solvents are used, such as methanol, ethanol, and water. Submonolayers form when the dye solution concentration is less than 10-4 M22-27,30 irrespective of the type of substrate, e.g., glass,23 quartz,24,27,30 organic crystals,25,26 and semiconductors.22,23,27 With this preparation scheme, almost all of the rhB molecules in the solutions were soaked up by the paper. We estimated the partition ratio of rhB molecules on the paper to those on the wafer to be ∼100:1. This high ratio is why we used a much higher concentration of the rhB solution than that used in previous studies,1-7,10-15 in which dye solutions of 10-8-10-10 M were dropped or spin-coated on a substrate surface. However, for convenience in our study, we simply refer to the prepared submonolayers as “10-7 M submonolayers” for example, on the basis of the concentration of the rhB solution used. The partition ratio was estimated by the following procedures: (i) The number of rhB molecules (No) before preparing adsorbates was computed from the volume (50 µL) and the concentration (10-4 M) of the rhB methanol solution. We kept the rhB solution from spilling from a Si wafer during preparation of a 10-4 M adsorbate; (ii) washing thoroughly the 10-4 M adsorbate with methanol and then evaluating the concentration of the washed solution by absorption spectroscopy; (iii) again the number of rhB molecules (Na) was computed from the volume and the concentration of the washed solution, and then the partition ratio (Na/No) was computed. We also obtained the equivalent partition ratio when using a 10-3 M adsorbate. Measurement of RhB Fluorescence in Submonolayers. Fluorescence was determined using a Nikon CF M Plan SLWD 100× objective (Obj, in Figure 1) with a numerical aperture (NA) of 0.75 and a working distance (WD) of 4.7 mm. For rhodamine fluorescence, we used a standard combination of a Nikon BA 580 long-pass filter (LP) and a Nikon DM 510 dichroic mirror (DM). The total area of a digitized image observable on the video monitor was 66.0 × 62.3 µm2, which was divided into 512 × 483 pixels when we used the 100× objective. Data acquisition time was 30 s at the maximum sensitivity of the photon-counting camera. A 100-mm focal length lens (FL) was used to obtain the average laser power density of ∼1.7 W/cm2 on the surface of a submonolayer and to obtain uniform irradiation across a field of view. From 5200 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
now on, we refer to the average power density only because the power density per pulse of the picosecond laser used here was below the intrinsic saturation power density of a rhB molecule.35 Photobleaching experiments were carried out to verify that the unitary photocounts measured from a single rhB molecule were the same for both the imaging and photobleaching experiments. To do this, the average power density used for the acquisition of fluorescence photocounts was tuned to ∼1.7 W/cm2, whereas the power density used for photobleaching was ∼2.4 W/cm2 to facilitate photobleaching as much as possible. We used the same experimental setup as that used in the imaging experiments, and selected fluorescent spots on the 10-5 M submonolayers. Photobleaching of rhB during the 30-s fluorescence acquisition period was negligible under the ∼1.7 W/cm2 excitation. The survival probability of rhB was more than 97%,36 assuming a photobleaching quantum efficiency (Φb) of 5 × 10-7, which is a typical Φb of several rhodamine derivatives in an aerated ethanol solution.37 The technical reasons why we chose the average laser power density (∼W/cm2) that was extremely lower than those used in previous studies7,9,11 (∼kW/cm2) are (i) intrinsic limitation of the output of the dye laser, (ii) necessity of irradiating as uniformly as possible a field of view under the 100× objective, and, more importantly, (iii) preventing the saturation of the photon-counting rate.38 Intense fluorescent spots that would most likely saturate the photon-counting rate or that might damage a photocathode were found occasionally in 10-4 M submonolayers under the ∼1.7 W/cm2 irradiation at the maximum sensitivity but rarely in 10-5 M submonolayers. To protect the photocathode, the photoncounting video camera automatically turned off when exposed to extremely intense light. We thus excluded such intense fluorescent spots from data acquisition to prevent the saturation mentioned in (iii). Another guideline in fluorescence measurements is to avoid strong scattering of the laser beam, which was visible to the naked eye on the Si wafer prepared on the X-Y-Z stage. Such strong scattering may be due to residuals from the cleaning procedure or lint coming from the surroundings after the cleaning was finished. Avoidance of strong laser scattering is effective in reducing background photocounts.39 Fluorescence spectra and lifetimes of individual fluorescent spots were simultaneously measured with a streak scope (Hamamat(35) The peak intensity of each picosecond pulse was 45 kW/cm2 per pulse, which was lower than the intrinsic saturation power density of rhB molecules, 180 kW/cm2. The saturation power density (Isat) was given by Isat ) hν/2στf, when σ ) 3.4 × 10-16 cm2 and τf ≈ 3.0 ns for rhB. (36) The estimated average excitation rate was ∼1600 photons/s for an irradiation of 1.7 W/cm2. Therefore, the survival lifetime from photobleaching (τb) for an irradiation of 1.7 W/cm2 was τb ) 1/(IavΦd) ) 1/(8.0 × 10-4 s-1) ) 1250 s. Thus, for this irradiation, the survival probability after 30 s was exp(-30/1250) ) 0.976. (37) Soper, S. A.; Nutter, H. L.; Keller, R. A.; Davis, L. M.; Shera, E. B. Photochem. Photobiol. 1993, 57, 972-977. (38) We maintained a maximum counts/frame per pixel of less than ∼0.1, or ∼90 counts/30 s per pixel, to avoid serious saturation in the maximum photosensitivity. With this criterion, the frequency of counting two photoelectrons as one photoelectron in each pixel during the frame time is lower than 5% of the number of correctly counted photoelectrons. (39) We obtained background photocounts of ∼330 counts/30 s per image (∼4.5 × 10-5 counts/s per pixel) including dark-current counts of ∼50 counts/30 s per image (∼6.7 × 10-6 counts/s per pixel) when irradiating a control wafer without adsorbed rhB with the ∼1.7 W/cm2 irradiation. We attributed the low background photocounts to the use of the photon-counting technique and to the optically clean surfaces of the Si wafers.
Table 1. Averaged Total Photocounts of an Image (66.0 × 62.3 µm2) versus rhB Concentration Used in Preparing Submonolayers concn (M) 10-4
1.5 × 1.5 × 10-5 1.5 × 10-6 1.5 × 10-7
photocounts/30 s per image 524130 [10] 51426 [15] 7720 [64] 976 [59]
a The total photocounts for each submonolayer varied among the images but stayed within a factor of ∼3 for each of the 10-6, 10-5, and 10-4 M submonolayers. In the 10-7 M submonolayers, this scatter increased because the number of fluorescent spots was low in each image. The total photocounts varied by more than a factor of 3, but remained within a factor of 15. The figures in the brackets are the number of images measured. Background photocounts (∼330 counts/ 30 s per image) are included in the above averages of the total photocounts. Dark-current noise (∼50 counts/30 s per image) that is intrinsic to the photon-counting video camera is included in the background photocounts.
Figure 2. Representative fluorescence images of rhB submonolayers prepared using various concentrations of rhB-methanol solutions: (A) 10-7, (B) 10-6, (C) 10-5, and (D) 10-4 M. The number of fluorescent spots for the respective concentrations were (A) 3, (B) 14, (C) 78, and (D) 117. The number of rhB molecules is assigned to the fluorescent spots in (A) and (B) according to the observations described in the Results section, Imaging of RhB Fluorescence in Submonolayers on Si Surfaces.
su C4334)40 coupled with a polychromator (Chromex 250IS, ) plus an optical microscope (Zeiss Axioplan) equipped with a photometer (Zeiss 2/UV) and a Nikon CF M Plan SLWD 100× objective. The 10-ps dye laser was also used for measuring fluorescence lifetimes. The streak scope without time sweeping (focus mode) was also used for measuring fluorescence spectra with a cw argon ion laser (514.5 nm). Preparation of Silicon Wafers with Oxide Layers of Known Thickness. Oxide layers of various thickness (260, 182, 98, 62, 43, or 20 Å) were prepared on the Si wafers. At the beginning we prepared an oxide layer thicker than 200 Å by thermal oxidation and then evaluated the thickness (260 Å). Following that, the 260-Å-thick layer was etched down to each thickness with a 1.6% HF aqueous solution. Each thickness was evaluated with an ellipsometer. Using Auger electron spectroscopy, we determined the thickness of the native oxide layer on the Si wafer to be 11 Å. Atomic Force Microscopy. Surface irregularities of the Si wafers were evaluated using a NanoScope III AFM (Digital Instruments) equipped with an n+-doped Si tip with a nominal spring constant of 56 N/m. The images were obtained using the tapping mode on an area of 512 × 512 pixels and with a scanning line speed of 1 Hz. RESULTS Imaging of RhB Fluorescence in Submonolayers on Si Surfaces. Fluorescence images of rhB submonolayers on Si surfaces were observed to determine the uniformity of Si surfaces. Representative images (Figure 2) of the four rhB concentrations (40) Ishikawa, M.; Watanabe, M.; Hayakawa, T.; Koishi, M. Anal. Chem. 1995, 67, 511-518.
(10-7, 10-6, 10-5, and 10-4 M) used for preparing submonolayers show three characteristics. First, the total photocounts summed over a full area of an image (66 × 62 µm2) showed near-linear dependence on the rhB concentration (Table 1). In determining this linearity, we avoided strongly emitting spots that probably saturated the photon-counting rate (see the Experimental Section, Measurement of RhB Fluorescence in Submonolayers). This linearity was a criterion for reproducibility in our fluorescence measurements involved in the imaging experiments and was evidence that the layers were true submonolayers. An appreciable departure from submonolayers is characterized by saturation of fluorescence intensity followed by a decrease in fluorescence intensity (due to dimer formation) with increasing dye concentration.22 Second, the number of bright spots also increased with increasing rhB concentration from 10-7 to 10-5 M, but saturated at a concentration between 10-5 and 10-4 M (Figure 2C and D). This means that a limited number of sites (pits or bumps) dotted on Si surfaces may allow rhB molecules to be adsorbed. In the section Comparison of Light-Scattering Sites with Fluorescent Spots, we discuss the relation between possible sites for adsorption and fluorescent spots. Third, fluorescence not associated with isolated spots became particularly strong when the rhB concentration exceeded 10-5 M. In the presentation of Figure 2, however, extra fluorescence not associated with isolated spots is visible only in the 10-4 M submonolayer (Figure 2D) because of the limited contrast of the presentation. This extra fluorescence was not due to contaminants in the solvent nor did it arise from the surroundings, but was from rhB itself because the total photocounts were proportional to the rhB concentration. Indeed, if the fluorescence photons not arising from the spots were due to contaminants, they should be uniform in all four pictures in Figure 2, irrespective of the rhB concentration. In the section Fluorescence Lifetime of RhB in Submonolayers on Si Surfaces, we discuss the origin of the fluorescence photons not associated with isolated spots. We also found that unitary photocounts of 100 or multiples of the unitary photocounts occurred in fluorescent spots irrespective of rhB concentration (10-7, 10-6, and 10-5 M). We summed the number of occurrences of photocounts from fluorescent spots (Figure 3A-C) using only photocounts for individual fluorescent spots and not for other background regions without fluorescent Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
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Table 2. Photocounts of Representative 20 Fluorescent Spots under the Irradiation of Vertically (V) and Horizontally (H) Polarized Excitationa V (counts/ 30 s)
H (counts/ 30 s)
51 64 73 75 88 95 97 100 101 107
6 10 11 10 10 7 17 8 12 20
V/H
V (counts/ 30 s)
H (counts/ 30 s)
V/H
8.50 6.40 6.64 7.50 8.80 13.6 5.71 12.5 8.42 5.35
112 112 112 143 154 160 160 204 207 235
16 19 14 14 29 13 16 26 30 29
7.00 5.89 8.00 10.2 5.31 12.3 10.0 7.85 6.90 8.10
a Both of the polarized lights were perpendicular to the incident direction: V-polarized light was in the x-z plane, and H-polarized light was perpendicular to the V-polarized light.
spots. The histograms show several maximums at every ∼100 photocounts, although each maximum had some variability in the photocounts. Thus, the approximate 100-count digitization strongly indicates that ∼100 photocounts come from a single rhB molecule under the conditions used here. All of the data in Figure 3A-C were obtained with the circularly polarized light. In contrast, the histogram in Figure 3D was obtained using vertically (V)-polarized light whose electric vector was perpendicular to the incident direction and was in the x-z plane (Figure 1). However, we were not able to make a histogram of photocounts using horizontally (H)-polarized light whose electric vector was perpendicular to the incident direction and to the V-polarized light because of a small number of the photocounts observed (Table 2). Two results further support our hypothesis that a single rhB molecule produces ∼100 photocounts: (i) stair-step photobleaching also occurred in representative fluorescent spots in units of unitary photocounts of 100 or multiples of the unitary photocounts (Figure 4) using the circularly polarized excitation, and (ii) the unitary photocounts of 100 were similar to the fluorescence photocounts from a single rhB molecule calculated using known experimental parameters.41 All of the fluorescent spots involved in Figures 2-4 were free from saturation of the photon-counting rate (i.e., ∼50 counts/30 s per pixel at the highest counts detected). Note again that ∼90 counts/30 s per pixel is equivalent to the 5% tolerance for counting two photoelectrons as one photoelectron.38 Fluorescence Spectroscopy of RhB in Submonolayers on Si Surfaces. To determine whether the rhB molecules were monomers in the prepared submonolayers, we measured fluorescence spectra of more than 50 individual fluorescent spots and background regions with no spots in the 10-5 and 10-4 M submonolayers. Because the number of fluorescent spots was low in the 10-7 and 10-6 M submonolayers, we selected 10-5 or 10-4 M submonolayers to simultaneously observe as many (41) A single rhB molecule absorbs ∼1600 photons/s or ∼48 000 photons during a 30-s acquisition time. Considering the photocathode efficiency at the fluorescence maximum (∼8%), the NA of the MCP (∼40%), the objective lens collection efficiency (∼17%), the net transparency of the objective lens used at the fluorescence maximum (∼60%),3 and the transparency of the DM used here (∼90%) and the LP (∼90%), we obtain ∼100 fluorescence photons during a 30-s acquisition time assuming Φf ) 1.
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Figure 3. Histograms of fluorescence photocounts evaluated from observed individual fluorescent spots versus the frequency of occurrences of the photocounts for (A) 10-7, (B) 10-6, (C) 10-5, and (D) 10-7 M submonolayers. Bin width is 20 counts. The histogram from 10-4 M submonolayers was not obtained because of difficulty in identifying fluorescent spots due to an increase in photocounts for background regions with no fluorescent spots. The data in (D) were obtained under experimental conditions different from those used in the data acquisition for (A-C): excitation of linearly polarized light (see text), the use of 532-nm light, and the reduced excitation power density (∼1.2 W/cm2) to balance the unitary photocounts (∼100 counts) in (A-C) with the unitary photocounts in (D).
fluorescent spots as possible within an image. For the 10-5 M submonolayers, we were not able to observe fluorescence spectra in the background region with no fluorescent spots (lower record in Figure 5E). This inability to observe fluorescence spectra in
Figure 4. Photobleaching kinetics of representative fluorescent spots on a 10-5 M submonolayer. Photocounts (Pc) of individual data points were obtained for 30-s accumulation periods with a power density of ∼1.7 W/cm2 using the circularly polarized excitation, for which a survival probability was higher than 97%.36 Photobleaching was induced with irradiation of ∼2.4 W/cm2. Error bars show ((Pc)1/2 for the individual data points. We observed high variability in the photobleaching kinetics among the experiments (i.e., fast or slow). (A) Example of fast photobleaching kinetics comparable to photobleaching kinetics in solution. The exponential curve shows the photobleaching kinetics for rhB in an aerated methanol solution for an irradiation power density of ∼2.4 W/cm2 and Φd ) 5 × 10-7.37 Recovery of the photocounts47 occurred at 55-60 min and at ∼70 min after the start of the experiment. The dotted line shows steps corresponding to increases and decreases in photocounts in units of unitary photocounts of 100 or multiples of the unitary photocounts. (B) An example of slow bleaching. The dotted line shows steps of the unitary photocounts of 100.
the background region is consistent with the fluorescence morphology of a 10-5 M submonolayer in Figure 2C, where fluorescence photons in the background region were more sparse than those in a 10-4 M submonolayer. All of the spectra from fluorescent spots were identified as those of rhB monomers, because the spectral shape and spectral maximum are similar to those of monomers observed in a dilute solution (Figure 5F). In contrast to the 10-5 M submonolayers, in the 10-4 M submonolayers, fluorescence spectra were observed from both fluorescent spots and regions with no spots and again were identified as those of rhB monomers. The fwhm of the spectra varied significantly from spectrum to spectrum in the 10-5 M submonolayers (Figure
5A-E), possibly reflecting a general feature that the number of rhB molecules entrapped within fluorescent spots in 10-5 M submonolayers is smaller than that in 10-4 M submonolayers and that spectral fluctuations6,11 of individual rhB molecules occur in 10-5 M submonolayers. Comparison of Light-Scattering Sites with Fluorescent Spots. We deduced from Figure 2 that the observed positions of fluorescent spots were associated with intrinsic sites (pits or bumps) on the Si wafers. One possible reason is that an increase in the number of fluorescent spots in the 10-7, 10-6, and 10-5 M submonolayers involves gradual filling of sites on a Si surface by rhB molecules. Furthermore, saturation of the number of fluorescent spots and appearance of the extra fluorescence not associated with fluorescent spots from the 10-5 to 10-4 M submonolayers are due to saturation of the sites and the subsequent spillover of rhB molecules not being able to settle within the sites. Thus, comparison between light-scattering images and fluorescent images were done to explore the possibility that fluorescent spots are located on the intrinsic sites of Si surfaces. We therefore selected 10-5 M submonolayers for further study because we expected, on the basis of Figure 2, a high correlation between the position of fluorescent spots and that of possible intrinsic sites. For the same field of view, we measured the surfaces of 10-5 M submonolayers under laser irradiation with and without the filters used for the fluorescence-imaging experiments. Without the filters, many spots (Figure 6A) formed by scattering of green laser light (light-scattering sites), and with the filters, fluorescent spots were observed (Figure 6B). The lightscattering sites observed in this study were isolated Airy disk patterns visible to the naked eye. This means that the size of a light-scattering site was smaller than ∼0.4 µm in diameter for the microscope we used. The position of light-scattering sites was strongly correlated with that of fluorescent spots, although the intensity of light-scattering sites was weakly correlated with that of fluorescent spots. These findings support our hypothesis that the position of fluorescent spots is correlated with the position of intrinsic sites on the Si wafers. However, although as much lightscattering contamination as possible is removed by the drop-anddrag treatment described in the Experimental Section, Preparation of Submonolayers, foreign particles from the surroundings may contribute to light-scattering sites. When using a Si wafer contaminated with foreign particles that we easily removed by wiping with a piece of lens-cleaning paper, we obtained a lower correlation than the previous comparison between fluorescent spots and light-scattering sites. This further supports our hypothesis. Physical Properties of Light-Scattering Sites. First, we examined light-scattering sites before and after drop-and-drag treatment without dye molecules using circularly polarized excitation. The observations are classified in three ways: (i) lightscattering sites that newly appeared after drop-and-drag treatment. However, we cannot identify whether they come from the paper or from other places on the wafer; (ii) light-scattering sites that disappeared after drop-and-drag treatments. They would not be pits. If they are pits, they should still be observed after the dropand-drag treatment. Nevertheless, we cannot conclude they are particles coming from the paper or surroundings because they Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
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Figure 5. (A-E) Fluorescence spectra of individual fluorescent spots in 10-5 M submonolayers observed with a streak camera in the focus mode and a cw 514.5-nm excitation. In (E), spectra from a spot (lower record) and from a background area not containing fluorescent spots (upper record). (F) Fluorescence spectrum of rhB in methanol (10-6 M) as a reference measured with the same fluorometer used for measuring spectra A-E.
could come from intrinsic bumps that were collapsed and dragged by the drop-and-drag treatment; (iii) light-scattering sites that were not removed with repeated drop-and-drag treatment (more than 10 times). Again we cannot conclude whether they are bumps or pits, although they are highly probable intrinsic scattering sites on Si wafers. Second, we examined the effects of polarized irradiation on the intensities of scattered light. We used linearly polarized light as we did in the imaging experiment (see the section, Imaging of RhB Fluorescence in Submonolayers on Si Surfaces) and observed two classes of scattering sites: one includes scattering sites that show stronger intensities when irradiated with the H-polarized light than the intensities obtained using the V-polarized light; the other includes scattering sites that show stronger intensities when irradiated with the V-polarized light than the intensities obtained using the H-polarized light. These observations show that each light-scattering site has some anisotropy for the polarization of the incident light. Fluorescence Lifetime of RhB in Submonolayers on Si Surfaces. The fluorescence showing isolated spots was welldefined in appearance, whereas the fluorescence not associated with the spots was diffused. The diffused fluorescence became particularly pronounced when the rhB concentration exceeded 10-5 M. This extra fluorescence came from rhB itself. 5204 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
To determine the origin of the diffused fluorescence and the fluorescence showing isolated spots, we measured the fluorescence lifetimes (τf) for both the fluorescent spots and regions with no fluorescent spots. We selected the 10-4 M submonolayers because both types of fluorescence were measurable (Figure 2D). The region with no fluorescent spots showed strong quenching (τf < 50 ps), as shown by the decay curve (a) in Figure 7A, whereas individual fluorescent spots showed τf typical for rhB (τf ) 1.0-2.7 ns), as shown in the decay curve (b). Figure 7B shows a histogram of τf for individual fluorescent spots, similar to τf previously measured in aqueous (τf ) 1.3 ns) or alcoholic (τf ) 2.0-2.7 ns) solutions.17-19 Our results identified strong quenching by Si wafers as an important factor in the diffused fluorescence. Our results show that the position of fluorescent spots strongly correlated with that of light-scattering sites and that the lightscattering sites were likely to be intrinsic sites on the Si wafers. From our expectation that the fluorescence in isolated fluorescent spots, or light-scattering sites, is insulated from quenching by an oxide layer, we studied the dependence of τf on the thickness (d) of oxide layers using the 10-4 M submonolayers. Figure 8 shows that τf increased with increasing d, finally reaching the limiting τf (2.6 ns) observed for a submonolayer on a quartz surface. Note that τf involved in Figure 8 was measured in bulk, thus reflecting the averaged nature of 10-4 M submonolayers.
Figure 6. (A) Light-scattering image obtained without the filters for rhB fluorescence on a 10-5 M submonolayer. Only spots from laser scattering were observed with the minimum sensitivity of the video camera. A 514.5-nm cw Ar ion laser beam was irradiated with the power density of ∼0.05 W/cm2. Red arrows show representative laser-scattering spots that coincide with the positions of fluorescent spots, and green arrows show representative light-scattering spots that do not coincide with the position of any fluorescent spot. (B) A fluorescent image observed in the same field of view as (A). The 514.5-nm beam with a power density of ∼5 W/cm2 was irradiated with the maximum sensitivity of the video camera. The red arrow shows a distinct fluorescent spot that does not coincide with the position of any laser-scattering spot. This result conclusively shows that rhB itself does not contribute to the laser-scattering spots. Note that the sensitivity used in (A) was ∼10 000 times higher than that used in (B) and that the laser power density used in (A) was ∼100 times higher than that used in (B). Selection of the spots was done using original images displayed on a video monitor, because rigorous comparison of laser-scattering spots with fluorescent spots was impossible in the two images presented here because of reduced contrast.
Irregularities of Si Surfaces. The surface irregularities (Figure 9) of the Si wafers used in the fluorescence measurements were mostly within (0.5 nm, as shown in the lower record of Figure 9. No sites that might correspond to the same kind of light-scattering sites shown in Figure 6A were found in the highresolution (500 × 500 nm2) AFM measurements. We searched Si surfaces for pits or bumps smaller than ∼0.4 µm on larger scanning areas (10 × 10 or 20 × 20 µm2) than the high-resolution measurement did, because they are possible for the lightscattering sites that were observed as diffraction-limited Airy disk patterns. Only bumps (typically 5-20 nm in height and 50-160 nm in diameter) were observed. The density of the bumps observed were ∼40/100 µm2 at the most. All of the bumps observed were not moved by repeated scans with the tapping mode. This observation denied the possibility that the bumps observed might be liquid or liquidlike materials. DISCUSSION We determined the number of rhB molecules in isolated fluorescent spots from the following evidence: (i) the digitized histograms in Figure 3A-C, (ii) the stair-step photobleaching in Figure 4, and (iii) the numerical consistency in the fluorescence photocounts from a single rhB molecule, as described in ref 41. The digitized histograms mean that rhB molecules remain independent in isolated fluorescent spots, within the limits of variability in photocounts around each maximum of the digitized histograms. The independent nature of individual rhB molecules may be surprising for researchers familiar with dye molecules, because dye molecules generally self-associate, particularly in a
concentrated aqueous solution.16 The independent nature is lost when dye molecules are strongly associated (e.g., form dimers). Kemnits and co-workers classified chemical species in submonoand monolayers of rhB as follows.24 Species I with fluorescence maximums in the 573-580-nm region is a monomer. Species II with maximums in the 590-600-nm region is a dimer. According to this classification, there were no dimers or higher aggregates of rhB in any fluorescence spectra observed in our study involving 10-7-10-4 M submonolayers. When preparing 10-3 and 10-2 M adsorbates, the coverage of which probably became mono- or mutilayers, we observed fluorescence spectra assignable to species II (dimers) only in 10-2 M adsorbates. Possible hydrogen bonds and ionic forces on the surfaces may prevent rhB molecules from forming dimers in the submonolayers. The shape of the histograms in Figure 3A-C is independent of rhB concentration (10-7, 10-6, and 10-5 M) over a concentration range of 2 orders of magnitude. This observation cannot be understood by assuming the surfaces involved here are physically and chemically uniform. If the surfaces were uniform, the shape of the histograms must change with increasing rhB concentration, according to Poisson distribution. The independent nature is consistent with the nonuniformity of the surfaces as revealed by the fluorescence images, the position-sensitive τf, and the lightscattering images. The histogram in Figure 3D shows again several maximums at every ∼100 photocounts, although three maximums near 300, 600, and 800 counts were biased toward lower counts and each maximum was blurred compared with the maximums in Figure Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
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Figure 7. (A) Representative fluorescence decay curves observed on a 10-4 submonolayer. A slow fluorescence decay curve (a), whose lifetime, τf ) 2.5 ns, was observed for an isolated fluorescent spot, whereas a fast decay curve (b), whose lifetime, τf < 50 ps, was observed in a region not containing isolated fluorescent spots. The upper record shows residuals between the fitting decay curve and the observed slow-decay curve. (B) Histogram of observed lifetime τf from individual fluorescent spots on 10-4 submonolayers.
Figure 8. Fluorescence lifetimes τf of rhB 10-4 submonolayers on Si wafers covered with an oxide layer of various thickness d (26.0, 18.2, 9.8, 6.2, 4.3, 2.0, and 1.1 nm). Solid line shows the theoretical curve from eq 1. Lifetime measurement involved with this figure was carried out in bulk without a microscope; thus, the observed lifetimes were averaged over an area larger than 100 × 100 µm2.
3A-C. It may be surprising that we found the digitized histogram, although disordered and blurred, using the V-polarized excitation light. We associate this observation with the ratio of photocounts determined by the V-polarized excitation to those determined by the H-polarized excitation (V/H in Table 2). From the high ratio of V/H (∼5-∼13), we consider that the transition dipole of a single rhB molecule is not parallel to a surface but is forced into a tilted position on the assumption that fluorescence intensity observed is entirely determined by the absorption efficiency. If 5206 Analytical Chemistry, Vol. 70, No. 24, December 15, 1998
Figure 9. An AFM image of a Si wafer used in the fluorescence measurements. No bumps or pits expected from the light-scattering images were observed in the high-resolution (500 × 500 nm2) scanning.
the transition dipole that is in a tilted position is completely immobilized on a surface within the acquisition time (30 s), the ratio of V/H should show a more sharp fluctuation than the fluctuation observed (within a factor of ∼3) from spot to spot. Thus, we consider a model that the transition dipole can rotate within a solid angle during the acquisition time. This model was proposed in the previous study.3 On the basis of this model, the transition dipole is proportional to cos θ along the z axis and (1/(21/2))sin θ along the y axis. Note that we canceled azimuth (φ) dependence by averaging from 0 to 2 π. Within the framework of this model, together with the assumption that absorption efficiency determines the observed fluorescence intensity and the known colatitude (∼65°) of the incident direction of the excitation light, we evaluated possible colatitude of the dipole to be 20-30° to reproduce the values of V/H in Table 2. Transition dipoles not parallel to a surface were concluded in the studies of submonolayers using surface second-harmonic generation21,28 and a recent single-molecule study.15 We observed strong fluorescence quenching in the region not containing isolated fluorescent spots. Here we consider possible mechanisms of this quenching. Three nonradiative processes should be considered. First, energy transfer among rhB molecules is not important as a mechanism of the strong quenching because of the low coverage of the submonolayers used here. No dimers were identified in the submonolayers involved in our study. An accepted mechanism of fluorescence quenching is monomer-to-dimer energy transfer, previously characterized by non-single-exponential fluorescence decays.20,22,24 However, single-exponential decays were only observed in our measurements involving 10-4 M submonolayers on quartz and on Si surfaces covered with oxide layers of various thickness. Second, quenching by electron transfer from rhB molecules to Si wafers is possible considering their relative
potentials.27,42 However, a possible electron-transfer rate between rhB molecules and a Si wafer is slower than the observed quenching rate even when rhB molecules come in direct contact with a Si surface.43 A 1.1-nm-thick oxide layer between rhB molecules and a Si surface existed even for the shortest τf on Si surfaces. Third, energy transfer is also possible between rhB molecules and Si wafers. This type of energy transfer was originally studied for dye-to-metal energy transfer. Cnossen and co-workers44 measured τf of rhB on an aluminum mirror covered with amylose-acetate ester of various thicknesses (1-6 nm). They used the following equation to analyze experimental observations, and found the equation to agree well with experimental data:
{
[ (
)
m(ω) - 1 η τf(d)-1 ) τf(∞)-1 1 + (dk)-3 2Im + 8 m(ω) + 1 6ξ
ωF ω 1 1 ω + 18 kFd ωp ωp ωp kFd
]}
(1)
where τf(d) is τf at a distance d from the mirror, τf(∞) is at an infinite distance from the mirror, k is the magnitude of the wavevector at an emitted frequency ω, m(ω) is the complex substrate (metal) dielectric constant, 1 is the dielectric constant of the medium in which the dipole is embedded, and h is an orientation parameter (h ) 3/2 for a perpendicular dipole and 3/4 for a parallel dipole). Here, kF is the Fermi wavevector, ωF the Fermi frequency, ωp the plasma frequency, and ξ ≈ 1 is a constant that depends on the electron-gas parameters. Only the first term in the brackets is important for our analysis, because the last two terms contain electron-gas parameters of metals. The first term represents the bulk contribution to the damping rate and is identical to the classical result where the interaction of the transition dipole with the electromagnetic field of its image dipole is considered.44 We thus computed τf using eq 1 (omitting the last two terms in the brackets) and plot the results in Figure 8 with a smooth line using τf(∞) ) 2.6 ns that was obtained on a quartz surface. A large deviation in the experimental data from the theory is notable below ∼15 nm. The reason for the deviation is not yet clear; however, the plot in Figure 8 is useful for a calibration curve of τf versus d of oxide layers. The plot thus gives important insight into d where fluorescent spots were observed, assuming that τf is determined only by d, and insight into which d we should select for analyte molecules to be free from quenching in analytical applications of Si wafers. We again carried out an imaging experiment using 10-7 M submonolayers on a 1700-Å-thick oxide layer on a Si wafer to inspect what happens to rhB fluorescence on the surface being (42) Laser, D.; Bard, A. J. J. Phys. Chem. 1976, 80, 459-466. (43) Sakata and co-workers29 reported an experimental and theoretical relationship between energy gap (∆E) and electron-transfer rate (ket) from photoexcited rhB to various semiconductors including Si. The energy gap is the difference between the photoexcited energy level of rhB molecules and the lowest edge of conduction bands of semiconductors. Using this relationship, together with the estimated energy gap ∆E ≈ 0.0 eV, we found that ket < 109 s-1. Therefore, the minimum fluorescence lifetime τf of rhB molecules in direct contact with a Si surface was estimated to be 0.72 ns using τf ) 2.60 ns obtained on a quartz surface and assuming ket )109 s-1. The estimated τf (0.72 ns) was longer than the τf (
