Letter pubs.acs.org/JPCL
Simultaneous Spectroscopic and Topographic Imaging of SingleMolecule Interfacial Electron-Transfer Reactivity and Local Nanoscale Environment Yufan He, Vishal Govind Rao, Jin Cao, and H. Peter Lu* Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, United States S Supporting Information *
ABSTRACT: The fundamental information related to the energy flow between molecules and substrate surfaces as a function of surface site geometry and molecular structure is critical for understanding interfacial electron-transfer (ET) dynamics. The inhomogeneous nanoscale molecule−surface and molecule−molecule interactions are presumably the origins of the complexity in interfacial ET dynamics; thus, identifying the environment of molecules at nanoscale is crucial. We have developed atomic force microscopy (AFM) correlated single-molecule fluorescence intensity/lifetime imaging microscopy (AFM-SMFLIM) capable of identifying and characterizing individual molecules distributed across the heterogeneous surface at the nanometer length scale. Using the novel AFM-SMFLIM imaging, we are able to obtain nanoscale morphology and interfacial ET dynamics at a single-molecule level. Moreover, the observed blinking behavior and lifetime of each molecule in combination with the topography of the environment at nanoscale provide the location of each molecule on the surface (TiO2 vs cover glass) at nanoscale and the coupling strength of each molecule with TiO2 nanoparticles.
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force, surface state density, and electronic coupling, based on single-molecule fluorescence spectroscopic measurements alone. Combining the single-molecule fluorescence spectroscopy approach with various other techniques such as computational studies,32,33 atomic force microscopy (AFM),29,34−36 electrochemistry,25,37−40 and Raman spectroscopy26,41,42 can facilitate inspection of multiple parameters with high chemical selectivity and wide temporal and spatial resolutions. Significant efforts made in this direction have already indicated the importance of various factors in determining injection dynamics and physical origins for interfacial ET rate fluctuations.29,34,38,41,43−47 Our earlier work on AFM correlated Raman spectroscopy has revealed inhomogeneous surface bonds, bonding energy, and vibrational relaxation energies at a single-TiO2 NP level which suggests that thermal-induced conformational motions of adsorbed molecules may contribute to fluctuations of electronic and vibrational couplings between the dye molecules and the TiO2 NPs.41 The results obtained indicate that interfacial ET rate fluctuations are caused by these energetic fluctuations. Additionally, in another work, we have shown that the single-molecule photonstamping spectroscopy correlated with electrochemistry provides novel insights for determining the nature of semiconductor energy states that participate in interfacial ET dynamics of porphyrin molecule anchored to TiO2 NP surface.25 Here, we have developed the
nterfacial electron transfer (ET) and energy transfer plays a critical role in surface chemistry, catalysis, and solar energy conversion, including solar photovoltaic and solar fuel science and technology.1−20 However, interfacial ET processes in dyesensitized semiconductor systems often involve complex and inhomogeneous dynamics, which comes from the spatial heterogeneities of the surfaces and the inhomogeneous coupling between the adsorbed molecules and the nanoscale rough surfaces of the substrates.21−27 The inhomogeneous interface makes it highly difficult for ensemble-averaged measurements to dissect the complex interfacial ET processes. The approach in single-molecule spectroscopy of studying one molecule at a time is a proven method to identify the inhomogeneity of the local environment. Our previous studies using single-molecule fluorescence spectroscopy and timecorrelated single-photon counting on dye molecules adsorbed on TiO2 nanoparticle (NP) surfaces demonstrate interfacial ET dynamics to be inhomogeneous and intermittent, and these intermittent dynamics are common for the interfacial chemical reactions that involve interaction between adsorbed molecules and substrate surfaces.21,24,28−31 The intermittency of ET dynamics is attributed to the perturbation and fluctuation of the vibronic coupling between the adsorbed dye molecules and the TiO2 surfaces. Although the expansion of single-molecule spectroscopy has allowed us to examine interfacial ET rate processes related to one molecule at a specific nanoscale site and at a time, it remains challenging to relate the observed intermittency of interfacial ET dynamics with the fundamental physical properties, such as the nanoscale environment, driving © XXXX American Chemical Society
Received: April 21, 2016 Accepted: May 23, 2016
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DOI: 10.1021/acs.jpclett.6b00862 J. Phys. Chem. Lett. 2016, 7, 2221−2227
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The Journal of Physical Chemistry Letters AFM correlated single-molecule fluorescence intensity/lifetime imaging microscopy (AFM-SMFLIM) to investigate individual molecules distributed across the heterogeneous surface to obtain nanoscale morphology as well as interfacial ET dynamics at the single-molecule level. Fluorescence lifetime is a measure of the time indicating how long a fluorophore remains in its excited state before returning to the ground state by radiative relaxations, which is a characteristic parameter of each fluorescent dye sensitive to molecular microsurrounding or conformational states. Typically, a fluorescence lifetime measurement can be used to probe the molecular environment or molecular conformational states.24,48,49 Single-molecule fluorescence lifetime imaging microscopy (SMFLIM) combines lifetime measurement with imaging: lifetimes obtained at the pixel-by-pixel in two dimensions are color-coded to produce images, which depend on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the concentration of the fluorophore. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information on its environment. Atomic force microscopy is a powerful approach that can provide surface and interface topographic images with nanoscale spatial resolution and is capable of identifying nanoscale spatial inhomogeneity.50−52 Therefore, a new approach of combining AFM and SMFLIM can provide not only the state information on a dye molecule and the environment information that surround the dye molecule, but also the morphology or surface structure information that surrounds the dye molecule. In this Letter, we report our newly developed approach of correlated AFM and single-molecule fluorescence intensity/lifetime imaging microscopy. Using AFM-SMFLIM, we investigate the mixed ((m-ZnTCPP/TiO2 NP) + mZnTCPP)/cover glass sample (Figure 1). We are able to identify the location of each dye on the surface (on top of TiO2 or on glass surface) at nanoscale as well as the relative coupling strength of each dye with TiO2 NPs. Experimental Setup of AFM-SMFLIM. The AFM-SMFLIM instrument is similar to and built based on our previously reported AFM fluorescence resonance energy transfer (AFM-
FRET) nanoscopy instrument.49,53 It consists of an inverted optical microscope (Axiovert-200, Zeiss) and an AFM scanning module (PicoSPM, Agilent) in an over−under configuration (Figure 1, left). The details of this AFM-SMFLIM system are described in the Supporting Information (Figure S1). A manual two-axis x−y mechanical positioning stage (Zeiss) and a computer-controlled two-axis closed-loop x−y 100 μm electropiezo-scanner stage (Madcity Instruments Inc.) were mounted directly onto the optical microscope. The two-axis closed-loop x−y electro-piezo-scanner stage was controlled by a computer with a raster scan software, by which we were able to scan the sample crossover laser in two dimensions to provide images and identify positions of single fluorescence molecule within the laser focus spot. The two-axis x−y mechanical positioning stage was used to support the closed-loop AFM scanning module (PicoSPM, Agilent). By adjusting the two-axis x−y mechanical positioning stage, we can move the AFM scanning module in two dimensions on top of the x−y electro-piezoscanning stage independently and set the AFM tip to a coaxis position with the laser beam. An AFM scanner was used to raster scan the sample to get topographic images. To prevent the AFM laser beam from interfering with the single-molecule fluorescence excitation laser and signal, the AFM scanning module was modified by a near-infrared superluminescent diode (SLD) laser at 950 nm to replace the conventional 650 nm laser source. Coaxial Alignment of Laser Focus with AFM Tip. Lining up the optical focal point and AFM tip is the first and critical step for a typical operation of our AFM-SMFLIM imaging analysis. First, we move the x−y two-axis mechanical positioning stage to place the AFM tip roughly coaxial with the laser beam focal point by observing the light reflection pattern from the AFM tip. A symmetric light reflection pattern can be observed from the microscope objective, which indicates that laser focus and AFM tip coaxial position alignment is achieved within a few micrometers. To coaxially align the AFM tip with the laser beam center of Gaussian distribution of the laser focus, we scan the AFM tip cross the area of the laser beam that has been aligned and send the APD signal to AFM controller through a gated photon counter SR400 (Stanford Instruments, CA), as shown in Figure S1. The obtained optical intensity image (Figure S2) shows a bright spot. This bright spot is due to the AFM tip, which reflects more photons as the AFM tip scans over the laser beam. Through this alignment procedure, we can align the AFM tip with the center of the laser beam. Single-Molecule Fluorescence Lifetime Measurement and Lifetime Image. Single-molecule fluorescence lifetime measurement and instrumentation have been reported in our previous publications.25,49 The excitation laser (532 nm, picosecond pulse, Coherent) beam was reflected by a diachronic beam splitter (z532rdc, Chroma Technology) and was focused by a highnumerical-aperture objective (1.3 NA, 100×, Zeiss) on the sample at a diffraction limited spot of ∼300 nm diameter. To obtain the fluorescence lifetime or lifetime images, each fluorescence photon’s arrival time, t, and the delay time between the laser excitation pulse and the fluorescence photon emission, τ (Figure S3), are recorded photon-by-photon by a PicoHarp 300 (PicoQuant) time-correlated single-photon counting (TCSPC) system. We use single-photon counting modules (SPCM-AQR-16, PerkinElmer Optoelectronics) utilizing a silicon avalanche photodiode (APD). Figure 2 shows the details for a typical two-dimensional (2D) fluorescence intensity image and fluorescence lifetime image. In
Figure 1. Left panel, scheme of AFM-SMFLIM imaging analysis; right panel, scheme of the sample, single m-ZnTCPP molecule coupled with TiO2 NPs, and single m-ZnTCPP molecule adsorbed on glass surface. 2222
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Furthermore, we can plot the histogram of the photon delay times in a binning period (20 ms) to obtain the fluorescence lifetime in each bin by fitting the histogram of delay time of all the photons in a 20 ms bin with exponential function or by calculating the mean of the delay time of all of the photons in 20 ms bins. We typically treat the photon counting distribution in each bin as a Poisson distribution that gives the means of each distribution as the fluorescence lifetime, τf. After calculating all the delay times in each 20 ms bin from the original photon stamping data (Figure 2A) and connecting the lifetime data resolved in all of the time bins, we obtain the fluorescence lifetime−time (τf vs t) trajectory; Figure 2C shows a period of fluorescence lifetime−time trajectory. We note that the total time for 2D image with a 100 pixels ×100 pixels matrix at 20 ms/pixel is 228 s, but not the 200 s from theoretical calculation. This 28 s difference between theoretical and real time is due to the scanner moving between pixels and between lines. Furthermore, as we two-dimensionally scan the sample over the laser beam, in addition to recording each photon by a TCSPC system (Picoharp 300, PicoQuant), we also record the time of each pixel in the 2D image scanning. Therefore, we can restore the 2D fluorescence intensity image (Figure 2D, by plotting the 2D matrix with each pixel’s photon counts) and 2D fluorescence lifetime images (Figure 2E, by plotting the 2D matrix with each pixel’s lifetime) based on the recorded photon stamping data (Figure 2A) and the time for each pixel in the 2D image scanning. Figure 3 show the fluorescence intensity image (Figure 3A) and fluorescence lifetime image (Figure 3B) of (m-ZnTCPP/ TiO2 + m-ZnTCPP)/cover glass; the image is 4 × 4 μm2 and 100 × 100 pixel with each pixel 20 ms. Figure 3C shows the correlated AFM image. From the fluorescence intensity image, there are about 10 molecules in the 4 × 4 μm2 area, in which three of them are apparently brighter than the other molecules. For convenience, we chose four molecules marked as 1, 2, 3, and 4 to present four typical situations. Molecules 1 and 3 appear bright, indicating high fluorescence emission; however, molecules 2 and 4 appear relatively darker than 1 and 3, indicating lower fluorescence emission. In Figure 3B, lifetime image, two of the molecules (1 and 2) show shorter lifetime; however, molecule 3 and 4 show the longer lifetime. From the correlation of the single-molecule fluorescence intensity image with single-molecule fluorescence lifetime image, it is evident that there is no direct relation between the fluorescence intensity and fluorescence lifetime. This further indicates that it is not necessary that brighter molecules will always have longer fluorescence lifetime and darker molecule will always have shorter fluorescence lifetime. To probe the environmental effects on fluorescence lifetime, we observe the correlated
Figure 2. Single-molecule photon stamping spectroscopic recording and analysis: a typical data trajectory (A), the intensity-time trajectory (B), and lifetime trajectory for a specific time period (C) which is deduced from panel A by calculating the mean value of the delay time of all the photons in each x−y scanning pixel time of a 20 ms bin. Fluorescence intensity image of (m-ZnTCPP/TiO2 + m-ZnTCPP)/ cover glass (D). Fluorescence lifetime image of (m-ZnTCPP/TiO2 + m-ZnTCPP)/cover glass (E).
this experiment, we scan the two-axis closed-loop x−y electropiezo-scanner stage with the sample by 4 × 4 μm2 area with 100 pixels × 100 pixels density of matrix at the rate of 20 ms/pixel. We also record the time matrix of each pixel in the 2D image scanning. Figure 2A shows the photon stamping data when the 2D electro-piezo-scanning stage scans the sample over the laser focus, in which each fluorescence photon registered with its chronic arrival time (t) and a delay time related to the picosecond laser pulse excitation (τ) (Figure S3). The chronic arrival times of the fluorescence photons contain the information about the photon flux so that we can count and bin the photons in a given time scale, for example, 20 ms binning time, to obtain a typical fluorescence intensity trajectory, as shown in Figure 2B. The high-intensity spikes reflect the laser scanning over a fluorescent molecule.
Figure 3. Correlated AFM−fluorescence intensity and lifetime data: fluorescence intensity image (A) and fluorescence lifetime image (B) of (mZnTCPP/TiO2 + m-ZnTCPP)/cover glass. The image size is 4 μm × 4 μm and 100 × 100 pixel with each pixel having 20 ms binning time. Correlated AFM image (4 μm × 4 μm) (C). 2223
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Figure 4. Typical single-molecule fluorescence intensity trajectory and respective fluorescence lifetime decay of (m-ZnTCPP/TiO2 + m-ZnTCPP)/ cover glass sample as marked in Figure 3: (A, E) molecule 1, (B, F) molecule 2, (C, G) molecule 3, and (D, H) molecule 4.
100−150 counts/10 ms). A similar phenomenon has been observed in our previous report21 and was attributed to the difference between the transition dipole direction of the individual ZnTCPP molecules and the laser polarization angle. However, Figure 4A,B shows strong fluctuations and blinking between bright and dark states, similar to our previously reported single-molecule fluorescence emission trajectories of m-ZnTCPP−TiO2,24 Coumarin 343−TiO2,21 pZnTCPP−TiO2,30 and PF−TiO2.28 These single-molecule fluorescence intensity fluctuations were attributed to the interfacial ET reactivity fluctuation of m-ZnTCPP on TiO2 NPs but not to the rotation or translational motions of the single molecules or the triplet state.24,30 The dark states in fluorescence intensity trajectories of m-ZnTCPP on TiO2 originate from the high ET reactivity, which quenches the S1−S0 radiative emission, and the bright states originate from the low ET reactivity, leaving the S1−S0 radiative emission dominant. Figure 4E−H shows the lifetime analysis data of the four marked molecules in Figure 3; panels G and H show longer lifetimes, and panels E and F show shorter lifetimes. It is clear that the lifetime data are correlated with the fluorescence intensity−time trajectories shown in Figure 4A−D. The
surface morphology by employing AFM. Figure 3C shows that two TiO2 nanoparticles are in the position where the fluorescence lifetime images show shorter lifetime, indicating that these two shorter fluorescence lifetime molecules are TiO2 NP coupled m-ZnTCPP molecules. For further understanding of microenvironment effects on the fluorescence intensity and fluorescence lifetime, we observe the single-molecule fluorescence fluctuation and fluorescence lifetime of each molecule. Figure 4 shows four typical singlemolecule fluorescence intensity trajectories and the respective fluorescence lifetime decays from four molecules that are marked 1, 2, 3, and 4 in Figure 3. It is clear that there are two types of single-molecule fluorescence emission trajectories. In Figure 4C,D, the single-molecule fluorescence emission trajectories show nearly constant levels of fluorescence intensity before photobleaching occurs. This observed behavior is similar to that of our previously reported single-molecule fluorescence trajectories of m-ZnTCPP,24 p-ZnTCPP,30 Coumarin 343,21 etc. on bare cover glass. Furthermore, Figure 4C,D shows apparently different intensity trajectories, under the same experimental conditions; the measured photon count in Figure 4D (about 20−30 counts/10 ms) is different from that in Figure 4C and that of the bright states in Figure 4A,B (about 2224
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The Journal of Physical Chemistry Letters fluorescence emission trajectories with a nearly constant level of fluorescence intensity show longer lifetimes, while the fluorescence emission trajectories with strong fluctuations and blinking between bright and dark states show shorter lifetimes. These observations along with the topographical AFM images clearly indicate that the fluorescence fluctuations and blinking arise because of ET from the excited state of the adsorbate mZnTCPP to the TiO2 NPs and fluctuating ET reactivity. The results derived from fluorescence intensity fluctuations and lifetime are well-correlated with Figure 3C, indicating that the ET reactivity and strong fluorescence intensity fluctuation or blinking occurred on TiO2 NPs and not on the cover glass surface. Therefore, these results identify the reactivity fluctuation and inhomogeneity of the interfacial ET dynamics. Additionally, the presence of relatively longer dark states for molecule 2 (Figure 4B) than molecule 1 (Figure 4A) suggests stronger coupling between molecule 2 and TiO2 than molecule 1 and TiO2. The stronger coupling between molecule 2 and TiO2 is further supported by shorter lifetime values (1.33 ns) observed in the case of molecule 2−TiO2 than molecule 1− TiO2 (1.88 ns). Therefore, AFM-SMFLIM can also be used as a tool to determine relative coupling strength between each single molecule and TiO2 NPs. Ef fect of AFM Tip on Fluorescence Lifetime.To understand the effect of the AFM tip on the imaged single-molecule fluorescence, we measured and compared fluorescence lifetime with and without the AFM tip. The results, as shown in Figure 5, indicate that the AFM tip does affect the fluorescence
electrochemically controlling the energetically accessible surface states of TiO2 NPs. We note that the Tahara group recently developed twodimensional fluorescence lifetime correlation spectroscopy, a combined fluorescence lifetime measurement with fluorescence correlation spectroscopy (FCS), which provides essential information about fast reaction dynamics in an equilibrated system at the single-molecule level.48 This method is particularly powerful for studying the microsecond scale conformational dynamics of biopolymers in dilute solution. Our current combined AFM-SMFLIM imaging can reach to the single-molecule level not only in air−solid interface but also in liquid−solid interface, which enables this imaging method to be used without the limitation of environment. The high versatility of the combined AFM-SMFLIM imaging suggests more extensive applications beyond just our current study on interfacial ET; for example, AFM-SMFLIM can be beneficial for many related research areas, including surface chemistry, electrochemistry, catalysis, energy transfer, and solar energy applications54,55 and even some biological processes, which can occur only in solution environment. The interfacial electron-transfer processes are often intrinsically complex, which comes from both spatial heterogeneity and the temporal inhomogeneity. The correlated AFMSMFLIM approach reported here is a preliminary step of responding to the call for both simultaneous spatial and temporal characterization of such complex and important interfacial electronic and energetic rate processes. While dynamic analysis will require femtosecond single-molecule spectroscopy, analysis of energetics of the process will entail analyzing the fluctuations and inhomogeneity of the driving force, electronic and vibrational coupling, solvent and vibrational relaxation energy, and local electronic work function at the single-molecule level with high spatial resolution, guided by different correlated AFM techniques. Hence, it is expected that combining single-molecule fluorescence spectroscopy approach with various other techniques such as AFM,29,34 electrochemistry,25,37−39 and Raman spectroscopy26,41 will provide more fundamental and profound understanding of the mostly inhomogeneous and complex interfacial ET dynamics related to catalysis, surface redox reaction dynamics, environmental sciences, and solar energy sciences. In conclusion, we have developed a new technical approach of AFM-SMFLIM, combining AFM and single-molecule fluorescence intensity/lifetime imaging microscopy, capable of resolving simultaneous single-molecule interfacial ET dynamics and local nanoscale topography for a targeted single molecule. The combined and correlated information about the fluorescence blinking behavior of each individual dye molecule and its lifetime along with the local nanoscale topography explains the inhomogeneity of coupling strength of each dye with TiO2 NPs and its effect on interfacial electron-transfer reactivity intermittency. The molecular-level understanding of the interfacial ET reactivity, derived from our study, sheds light on the intrinsic fluctuating and inhomogeneous interfacial ET dynamics, which may, for example, help in the development of solar energy conversion science and photocatalysis.
Figure 5. Histograms derived from single-molecule fluorescence lifetimes of (m-ZnTCPP/TiO2 + m-ZnTCPP)/cover glass sample without AFM tip perturbation (A) and with AFM tip perturbation (B).
lifetime of single molecules. A histogram of single-molecule fluorescence lifetime resulting from more than 100 molecules without AFM tip perturbation indicates that single-molecule fluorescence lifetime distributed in two main regions, with peaks at 1.8 and 4.8 ns and mean lifetime value of 3.65 ± 1.66 ns. The two lifetimes essentially correlate well with the mZnTCPP in two kinds of environments, m-ZnTCPP on TiO2 NPs and on glass surface. However, with AFM tip perturbation, the measured lifetimes (Figure 5B) are mostly shorter, having mean lifetime value of 3.09 ± 1.69 ns, because of the quenching of the excited state of the adsorbate, m-ZnTCPP, by the metalcoated AFM tip. Nevertheless, the AFM-SMFLIM imaging provides a direct characterization of the correlation between single-molecule interfacial ET reactivity and its nanoscale local environment. Additionally, we can also use the metallic AFM tip to apply electric potential to each single molecule without affecting other molecules, which is desirable and presumably powerful for dissecting complex interfacial ET dynamics by probing each single molecule anchored to a TiO2 NP surface while
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EXPERIMENTAL SECTION Materials and Sample Preparation. Zn(II)-5,10,15,20-tetra(3carboxyphenyl) porphyrin, m-ZnTCPP, and dichloromethane were purchased from Frontier Scientific (research and development purpose only) and EMD chemicals (HPLC grade), 2225
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The Journal of Physical Chemistry Letters respectively. Ethanol (200 proof; anhydrous, ≥99.5%) and poly(methyl methacrylate) (PMMA; MW 15 000 g mol−1) were purchased from Aldrich. All the reagents were used as received. Cover glasses (Fisher; 18 × 18 mm2; thickness, 170 μm) were thoroughly cleaned by sonication in deionized water, ethanol, acetone (CHROMASOLV Plus, for HPLC; ≥99.5%; Aldrich), and deionized water, each for 20 min, and then dried using nitrogen gas before their use. Nanometer-sized TiO2 particles were prepared by the hydrolysis of titanium isopropoxide (Aldrich, 99.999%) as precursor according to a literature protocol.56 The size range (diameter) of the TiO2 nanoparticles was found to be 5−15 nm, as determined by atomic force microscopy. For the single-molecule experiments, we prepared the sample following our reported procedures.21 Briefly, the sample of m-ZnTCPP/TiO2 NPs + m-ZnTCPP on clean glass (as described in the scheme shown in the right-hand panel of Figure 1) for interfacial ET study was prepared by first spin coating 25 μL of 0.1 nM m-ZnTCPP/TiO2 NP solution on a clean cover glass at 3000 rpm followed by spin coating 25 μL of 0.1 nM m-ZnTCPP in ethanol and 50 μL of PMMA (in CH2Cl2, 1 mg/mL) at 3000 rpm. The PMMA thin film is used to protect the dye molecules from singlet O2 photobleaching.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b00862. Experimental setup of single-molecule AFM-fluorescence microscopy, schematic diagram of coaxial laser and AFM tip, and photon stamping concept and definition of the parameters (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is supported by the US Department of Energy (DOE), Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences & Biosciences.
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REFERENCES
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Letter
The Journal of Physical Chemistry Letters
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DOI: 10.1021/acs.jpclett.6b00862 J. Phys. Chem. Lett. 2016, 7, 2221−2227
