Article pubs.acs.org/JPCC
Single Molecule Wobbling in Cylindrical Mesopores Rajib Pramanik, Takashi Ito,* and Daniel A. Higgins* Department of Chemistry, Kansas State University, Manhattan, Kansas 66506-0401, United States S Supporting Information *
ABSTRACT: Simultaneous single molecule tracking (SMT) and single molecule emission dichroism (SMED) measurements are used to investigate the translational and orientational motions of fluorescent probes diffusing within mesoporous silica films. The results afford a quantitative measure of molecular confinement within the mesopores and allow for the accessible pore diameter to be determined with higher precision than can be achieved by common SMT methods alone. In these studies, dual-polarization wide-field video microscopy is used to obtain SMT data simultaneously in two orthogonal polarizations. A rod-shaped perylene diimide dye (C11OPDI) is employed as the probe and is excited by circularly polarized light. SMT reveals a predominance of one-dimensional dye diffusion within the mesopores. The SMED data demonstrate that the molecules diffuse in an oriented state, with their long axes parallel, on average, to the long axis of the mesopores. Theoretical expressions quantitatively relating the SMED and SMT results to the extent of C11OPDI orientational wobbling are developed. The results are consistent with confined wobbling of the single molecules within the mesopores. An ensemble-averaged wobbling angle of 19 ± 3° is obtained, yielding an estimated accessible pore diameter of 1.3 ± 0.2 nm.
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characterizing their orientational mobilities. The first fluorescence-based single molecule orientation measurements were made using the structured optical fields of a near-field optical probe.13 Since that time, a number of far-field polarization modulation14−16 and emission-pattern imaging17−20 methods have been reported. These have largely been used to observe milliseconds and longer single molecule orientation fluctuations. Fluorescence dichroism (or anisotropy) data14,15,21−23 provide orientation information on these same time scales but also afford a route to assessing much faster orientational motions through depolarization of the detected emission. In these studies, excitation may employ linear or circular polarization, or unpolarized light, while detection in two orthogonal polarizations is used to access dichroism data. The measured dichroism in studies of confined single molecule motions depends upon two parameters: (1) the average orientation of the molecule relative to the detected polarizations and (2) its orientational dynamics (e.g., wobbling motions) on relevant time scales. Several methods for exploring the restricted tilting and wobbling motions of dye-labeled biomolecules have already been established.24 These frequently require multiple measurements using different excitation and emission polarizations. When molecular motions confined within 1D nanostructured materials are being investigated, simpler methods that combine SMT and single molecule emission dichroism (SMED) measurements25 can be employed. Together, these provide quantitative data on both
INTRODUCTION One of the most exciting applications of single molecule detection is in the characterization of confinement effects within nanostructured materials. Single molecule tracking (SMT) methods provide a valuable route to detecting the hindered translational motions of probe species in such systems.1 Some of the most dramatic visual evidence of confined molecular diffusion can be found in recent SMT studies of mesoporous silica,2−7 block copolymer films,8,9 microporous coordination polymers,10 and lyotropic liquid crystalline mesophases.11 The molecules tracked in these studies were found to move predominantly along one dimension (1D), showing that their motions are guided by the cylindrical nanochannels or nanodomains incorporated within the materials. Beyond these anticipated confinement effects, direct evidence for blockage of probe motion along the nanostructures has also been obtained.7 While SMT provides good evidence of hindered translational motions, confinement should also have a profound effect on the orientational mobility of probe molecules. The orientational confinement of molecules is again readily demonstrated in materials comprised of nanometer-sized cylindrical channels, such as mesoporous silica.2,3,5,12 Many SMT studies have shown that rod-shaped probes such as the perylene5 and terylene12 diimides diffuse in a highly oriented state, with their long axes oriented along the pore axis. While these observations provide qualitative evidence of orientational confinement, valuable quantitative data have not yet been reported to our knowledge. Significant effort has been devoted to the development of methods for measuring single molecule orientations and for © 2013 American Chemical Society
Received: January 15, 2013 Revised: January 23, 2013 Published: January 23, 2013 3668
dx.doi.org/10.1021/jp400479w | J. Phys. Chem. C 2013, 117, 3668−3673
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to yield improved precision over SMT-based superlocalization results under equivalent experimental conditions.
average molecular orientation and the extent of orientational wobbling. Data on molecular wobbling can provide additional quantitative information on relevant feature sizes in 1D nanostructured materials. In this work, polarization-dependent wide-field single molecule fluorescence imaging25 is employed to simultaneously obtain SMT and SMED data from rod-shaped perylene diimide molecules (C11OPDI, see Scheme 1) diffusing within the
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EXPERIMENTAL CONSIDERATIONS Silica films incorporating locally aligned cylindrical surfactantfilled mesopores were obtained by spin-coating surfactanttemplated silica sols onto glass coverslips.5 The detailed procedures employed are described in the Supporting Information. Briefly, tetramethoxysilane was employed as the silica precursor, and cetyltrimethylammonium bromide (CTAB) was used as the structure-directing agent. The silica films were doped to nanomolar concentrations with N,N′bis(octyloxypropyl)perylene-3,4,9,10-tetracarboxylic diimide (C11OPDI, Scheme 1). All SMT experiments were performed under a mixture of water/ethanol vapor. Exposure to solvent vapor is required to complete the formation of the cylindrical mesopores26 and to mobilize the dye molecules for SMT studies.27,28 The optical configuration employed for recording SMED and SMT data is illustrated in Scheme 2. The microscope and general SMT procedures have been described previously in detail.5 Briefly, C11OPDI fluorescence was excited with 488 nm laser light. A quarter-wave plate was used to obtain (nominally) circularly polarized light. The light was subsequently passed through a spinning optical diffuser, reflected from a dichroic beamsplitter (Chroma, 505 DCLP) and focused into the back aperture of an oil immersion objective (Nikon, Apo TIRF 100×, 1.49 numerical aperture, NA). The incident power was maintained at ∼0.75 mW. Fluorescence from the sample was collected by the same objective and directed back through the dichroic beamsplitter, through a bandpass filter (Chroma, HQ535/50m) and into an image splitter (Cairn Research, Optosplit II) incorporating a polarizing beamsplitter cube. Two separate images of the field of view were produced, in which the collected light was polarized along orthogonal directions. The two polarization states are arbitrarily defined as horizontal (H) and vertical (V), based on their projections in the image plane. Single molecule emission was detected by a back-illuminated electron multiplying (EM) CCD camera (Andor, iXon DU897). All videos were acquired using an EM gain of 30 and 2 × 2 on-chip binning, giving a calibrated pixel size of 125 nm. Videos were typically acquired at frame rates of ∼3 frames/s for a total length of 200 frames.
Scheme 1. Pictorial Representation of C11OPDI Wobbling in a CTAB Templated Cylindrical Silica Mesopore
cylindrical pores of mesoporous silica films. These experiments provide quantitative data on the confined translational and orientational motions of the molecules. A model for wobbling of the dye within the mesopores is shown in Scheme 1. Circularly polarized incident light is employed for nonselective single molecule excitation. SMT and SMED data are obtained by splitting the collected fluorescence into orthogonally polarized images, which are simultaneously recorded using a single CCD camera (see Scheme 2). Theoretical expressions Scheme 2. Diagram of the Polarization Selective Wide-Field Fluorescence Microscopea
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a
RESULTS AND DISCUSSION A representative video frame is shown in Figure 1a. The video itself is provided as Supporting Information (video 1). Slowly diffusing C11OPDI molecules produced round, diffractionlimited fluorescent spots. These comprise the majority of spots analyzed. Rapidly diffusing molecules produce elongated streaks that vary dramatically in appearance, depending on the exact motions exhibited by the molecule during each frame. SMT and SMED measurements begin by fitting these spots to 2D Gaussian profiles, although the fitting is inexact for rapidly moving molecules. The fitting procedure yields the location of each molecule, the amplitude of its emission profile, the width of the spot it produces, and the background signal level. Spot fitting was facilitated by convolving all images with a Gaussian function having σ = 1 pixel and by use of predetermined X,Y pixel offsets for the spot pairs. These procedures were required for locating the very weak emission profiles produced by molecules oriented approximately perpendicular to either one
The sample is illuminated with circularly polarized light.
are developed to quantitatively determine the maximum wobbling angle for each molecule. The model accounts for differences in the average orientation of each molecule with respect to the polarization axes and for depolarization caused by the high numerical aperture objective. The results show that wobbling of C11OPDI is confined to ∼±19° from the mesopore axis, consistent with a mean accessible pore diameter of ∼1.3 nm. The use of simultaneously recorded SMED and SMT data for pore size determinations in cylindrical nanopores is shown 3669
dx.doi.org/10.1021/jp400479w | J. Phys. Chem. C 2013, 117, 3668−3673
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pores is strictly unknown, X-ray scattering results indicate the average pore spacing is 4.3 nm.5 These data suggest the surfactant filled pores have a diameter of ∼4 nm.30 The pore size is also estimated to be ∼3.8 nm from the fully extended conformation of CTAB. Similarly, the full length of C11OPDI is estimated to be ∼4.1 nm. The wobbling angle exhibited by the molecules can be quantitatively determined by carefully analyzing the emission intensity of each molecule in the pairs of orthogonally polarized images. The model employed is presented in Supporting Information. The final equations defining the dependence of the signal on wobbling-induced depolarization are given in eqs 1 and 2: IV̅ ∝ cos2 ϕ(1 − cos3 θmax ) +
1 2 (a + sin 2 ϕ) 2
× (2 − 2 cos θmax − cos θmax sin 2 θmax ) IH̅ ∝ sin 2 ϕ(1 − cos3 θmax ) + Figure 1. Representative images of C11OPDI in CTAB templated mesoporous silica obtained simultaneously for a single sample region in two orthogonal polarizations. Double-ended arrows designate the detected polarizations. (a) Images depicting a single frame from the video provided in Supporting Information (video 1). (b) Images showing the 1D motions of the individual molecules. The latter were obtained by plotting the maximum signal at each pixel across the final 100 frames of the video. The single-ended arrows point to molecules used in Figures 2a,b and 3. The left and right panels in (a) and (b) are displayed on identical intensity scales.
(1)
1 2 (a + cos2 ϕ) 2
× (2 − 2 cos θmax − cos θmax sin 2 θmax )
(2)
Here, IV̅ and IH̅ represent the emission intensities measured from the pair of spots produced by each molecule in the vertical and horizontal polarization channels, respectively. The maximum wobbling angle is defined by θmax (see Scheme 1). Two important factors have been accounted for in developing these equations. First, the signal observed is strongly dependent on the average orientation, ϕ, of each molecule, relative to the detected polarizations (measured from vertical on the images). The average orientation is determined from the trajectory angle for individual 1D diffusing molecules (see below). Second, the use of a high NA objective causes some emission depolarization. This effect is accounted for in the a2 parameter, which is estimated from a previously published model15 to be ∼0.44 for the 1.49 NA objective employed. While the dichroic mirror in the microscope alters the excitation polarization,31 this effect only leads to slight variations in the detected intensity with average orientation. While the emission polarization is also be altered by the dichroic mirror, the detection efficiency at the emission wavelength was found to be identical for the two polarization channels to 100 nm length scales suggests this is unlikely. However, investigations of the wobbling angle as a function of probe molecule length are now being performed to verify the origins of the depolarization. Preliminary analysis of the results indicates that wobbling of the molecules within the pores is the correct interpretation. These results will be reported in a subsequent paper. It should be noted that the accessible pore diameter could theoretically be determined by well-known SMT-based superlocalization methods. While single molecule localization precision as small as 1.5 nm has been reported,34 this generally requires the use of oxygen scavengers and/or triplet quenchers or relatively high incident powers and may not be achievable in all samples. For example, the best localization precision reported to date for single molecule studies of mesoporous silica is ∼2−3 nm.35 The Fisher information limit36 to the localization precision in the present experiments is ∼1.2 nm (535 nm emission, 1.49 NA objective, ∼2200 photons detected). Estimates using the method of Webb and coworkers37 yield a value of ∼6 nm (including background noise of ∼12 counts, a pixel size of 125 nm, and a point spread function width of 150 nm). The mean localization precision in
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CONCLUSIONS In conclusion, we have established a polarization-dependent wide-field SMT method for simultaneously probing the confined translational and orientational motions of single molecules diffusing through 1D mesopores. The theory necessary for quantitative interpretation of the SMT and SMED data in terms of the molecular wobbling angle was presented. Analysis of the experimental results show that the orientational motions of single C11OPDI molecules diffusing through the surfactant-filled pores of mesoporous silica films are strongly confined, exhibiting an ensemble-averaged maximum wobbling angle of ∼19°, consistent with a ∼1.3 nm accessible pore diameter. The use of single molecule wobbling angle data for determination of the accessible pore size in cylindrical nanopores was shown to afford higher precision than superlocalization results derived from the same videos. The method and theory reported herein will afford new routes to a deeper understanding of mass transport and molecular confinement within mesoporous materials being developed for improved chemical separations.
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ASSOCIATED CONTENT
S Supporting Information *
Additional details on sample preparation, characterization, and data analysis along with a representative video. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (T.I.);
[email protected] (D.A.H.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge support from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of 3672
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Basic Energy Sciences of the U.S. Department of Energy (DEFG02-12ER16095). Khanh-Hoa Tran-Ba and Seok Chan Park are acknowledged for their help with sample preparation and data collection.
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