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Optical imaging and tracking of single molecules in ultrahigh vacuum

Molecule-surface interaction is key to many physical and chemical processes at interfaces. Here, we show that the dynamics of single molecules on a surface under ultrahigh vacuum can be resolved using fluorescence imaging. By adapting oil-immersion microscopy to a thin vacuum window, we measure the surface adsorption, translational and rotational diffusion of single perylene molecules on a fused silica surface with high spatial and temporal resolutions. Time-dependent measurements of the fluorescence signal allow us to deduce two characteristic decay time scales, which can be explained through a simplified model involving two adsorption states and five energy levels. The system presented in this work combines fluorescence imaging with essential ingredients for surface science and promises a platform for probing single molecule-surface interactions in highly defined conditions.


Introduction
Physical and chemical processes at interfaces are fundamental to a wide range of applications covering surface catalysis, 1 optoelectronics, 2 life science, 3 and quantum technologies. 4,5 The ability to resolve individual molecules and track their dynamics on well-defined surfaces is crucial for the understanding of these processes. Motivated by these interests, moleculesurface interaction has been studied in different scientific contexts and employing different technologies. Scanning probe techniques such as atomic force microscopy 6,7 and scanning tunneling microscopy 8,9 are proven tools for imaging and manipulating single molecules on surfaces with spatial resolutions down to the atomic scale. Harnessing the nanocavity plasmon modes supported by the tip-substrate geometry, scanning probe techniques can be further combined with laser excitation, allowing optical microscopy and spectroscopy through the detection of fluorescence 10,11 or tip enhanced Raman scattering. 10 single-molecule localization methods, 22 fluorescence imaging allows wide-field visualization of molecular diffusions with simultaneously high temporal and spatial resolutions. 23 While optical microscopy of single molecules is widely applied in studying dynamic processes, a prerequisite is efficient photon collection. 24 The fluorescence signal from a single molecule in a detection time window is ultimately limited by its excited-state lifetime. In applications under ambient conditions, efficient photon collection is commonly achieved through the use of oil-immersion objectives with high numerical apertures (NA). As a result, spatial localization precision of ∼10 nm and time resolution of 1000 frames per second are within reach. 22,23 Implementing similar constructions in UHV settings is challenging due to the outgassing of the immersion oil and the imaging optics. Moreover, typical high-NA objectives have working distances in the order of 100 µm, leading to increased complexities in positioning and vibration control inside the UHV chamber. These challenges can be circumvented by using a thin vacuum window, that allows a high-NA objective to image its interior surface.
In the past decades, various types of thin windows have been reported for specialized applications. Imaging single molecules on the inner surface of a thin window was employed for spatially-resolved detection of matter-wave diffraction . 25,26 Thin windows were also used for electron microscopy of samples in air and liquid 27,28 and imaging cold atoms in an optical lattice . 29 Here, we show that single-molecule dynamics on a surface under UHV can be studied with spatial and temporal resolutions at the same level with experiments under ambient conditions. This is enabled by the combination of oil-immersion microscopy with a homemade 150 µm-thin vacuum window.

Experimental setup
To fabricate a vacuum window suited for oil-immersion microscopy, we start with a 4 mmthick fused-silica window plate, and drill a 2 mm-diameter hole through its center using a diamond-coated drill head, as illustrated in Fig. 1(a). The window is then cleaned in acetone, methanol, piranha consecutively and rinsed with ultra-pure water. A 150 µm-thin fused silica coverslip, cleaned through the same procedure, is brought into optical contact with the window through van der Waals bonding , 30 with its center matched to the hole on the window. The assembly is then transferred into a muffle furnace, annealed at 950°C, and under atmospheric pressure for about 8 hours. During this process, covalent bonds are developed between the two surfaces 31 and the resulting piece becomes vacuum tight.
The window is then installed on a demountable Conflat viewport (VPD-1.19, Accu-Glass Products Inc.), with the bonded coverslip facing the atmospheric side [see Fig. 1 Low-autofluorescence immersion oil is applied between the objective and the coverslip, as displayed in Fig. 1 The light emitted by a diode laser at 405 nm is used to illuminate the inner surface of the coverslip. The laser is first coupled out from a fiber coupler, spectrally cleaned by a bandpass filter (HC Laser Clean-up MaxDiode 405/10, IDEX/Semrock) and sent through a polarization beamsplitter and a pair of half-and quarter-waveplates. Before entering the objective, a f = 250 mm lens focuses the light to achieve a wide-field illumination, see Oil-immersion microscopy of single molecules

Translational and rotational diffusion
Having the capability to image single adsorbed molecules, we turn to investigate their translational diffusion on the fused silica surface. To do so, we first identify individual molecules in each frame using the software TrackMate. 35 For each identified molecule, a maximum likelihood estimation procedure is applied with the point-spread function (PSF) of the system as the model function. The resulting uncertainty of the center position yields the localization precision. 22 With 20 ms exposure time on the camera, we achieve an averaged localization precision of ±24 nm. Localized single molecules in consecutive frames are linked based on their mutual distances. 36 Figure 3(a) displays the trajectories of 120 molecules identified during two seconds of observation time. We find that the majority of the molecules showing persistent fluorescence signal are strongly confined in position, as indicated by the black trajectories. These molecules are likely tightly bound to their local adsorption sites, and their coupling to the surface quenches the long-lived internal states efficiently, yieldings stable fluorescence signals. In contrast, we also identified molecules with significantly higher mobility, as indicated by the blue trajectories in Fig. 3(a). These molecules show reduced fluorescence stability and shorter residence times on the surface. Figure 3 In addition to the translational diffusion, the rotational diffusion of single molecules on the surface can be tracked through polarization anisotropy measurements . 37 We limit our studies to the two-dimensional (2D) rotational diffusion 38 by deducing the 2D fluorescence polarization anisotropy r = (I h − I v )/(I h + I v ) for each molecule. Here, I h,v denote intensities of the horizontally and vertically polarized fluorescence light measured by the camera, respectively. The molecules are illuminated with circularly polarized light by adjusting the half-and quarter waveplates [see Fig. 1(c)]. To access these two polarization components, a Wollaston prism (WPM10, Thorlabs Inc.) is placed before the imaging lens [L2 in Fig.1(c) where the molecule enters a dark state such as the triplet or charge-separated state. 34,39 Photoblinking can last for milliseconds due to the lack of quenching agents in oxygen-free conditions . 40,41 Using the fluorescence signal, we reconstruct the transition dipole orientation θ of the molecules through the relation tan 2 θ = I v /I h . The results are displayed in Fig 4(d).
Here, we forced the angle to fall into the range of [0, π/2]. The discontinuities in the angular trajectories are results of photoblinking. Similar to the observations in translational diffusion, we observed molecules with strong angular confinement, as exemplified by the red curve. Molecules of this type are likely undergoing 'wobbling-in-cone' type of rotation . 42 At the same time, we also observed molecules with higher rotational mobility, as indicated by the blue and green curves.
This set of measurements were performed with an exposure time of 10 ms for each frame.
Using this setting, an average uncertainty of ±8°in determining θ is achieved. The main contributions to the uncertainty come from photon shot noise and fluctuations of the fluorescence background. We also note that the polarization contrast of the setup is limited by transmission through the high-NA objective to the level of 15:1. The detection optical path features a slight dichroism, which amounts to about 3% difference in transmission for the two orthogonal polarizations. This systematic offset is corrected in the data processing.
The inhomogeneity in the diffusional behaviors points to the co-existence of different adsorption states of perylene molecules on the surface. This may not be surprising considering the size of the molecule and the complexity of the amorphous fused silica surface. In early studies, it was identified that the freely vibrating hydroxyl groups are mainly responsible for the adsorption of smaller aromatic hydrocarbons such as benzene. 43 However, other possible ending groups such as siloxane, and hydrogenbonded hydroxyl groups also affect the adsorption. 43 In addition, surface defects could also introduce strong modifications of local potentials and lead to strong bindings.

Temporal dynamics of fluorescence signal
To gain further insight into this system, we measure the temporal dynamics of the ensemble fluorescence intensity under constant laser illumination. The measurement starts with the ablation of molecules from the target using a single laser pulse. After the ablation, the incidence of molecules on the fused silica window is measured through the change of fluorescence intensity between subsequent camera frames, as displayed in Fig. 5(a). The peak between 0.15 s and 0.25 s shows the time of arrival of molecules. The insets display a sequence of images acquired at different times before and after the influx of molecules. After the ablation, the window is continuously illuminated by the detection laser. Rapid decay of the fluorescence intensity is observed, as shown by the solid black curve in Fig. 5(b). At longer time scales, the fluorescence intensity approaches the background level, as indicated by the last image in the inset of Fig. 5(a). A detailed observation of the decay signal reveals its non-mono-exponential character. Non-exponential decays can result from the heterogeneity in adsorption or internal states of molecules, 34 or due to nonuniform illumination profiles. 44 The influence of the latter is minimized by micro-sectioning the camera images to areas much smaller than the illumination profile.
The signal could be fit with a biexponential function, as shown by the dashed red line in  Fig. 5(b), for each adsorption state, we consider the optical transitions between the singlet ground state S 0 (S 0 ) to a singlet excited state S 1 (S 1 ). Once a molecule is excited, it either returns to the ground state or reaches a dark state L through photobleaching or desorption. We note that for both mechanisms there can be intermediate states involved. Here, for the sake of simplicity, we describe the contribution of all the pathways with an effective rate γ (γ ). The rate of laser excitation and that of the decay via S 1 → S 0 (S 1 → S 0 ) are described by p (p ) and f (f ), respectively. The kinetics of the system can be described with rate equations based on this model. Knowing that the timescales of optical excitation and fluorescence decay are significantly shorter than that of photobleaching and desorption, we apply the steady-state approximation to the rate equa-tions associated with S 0,1 (S 0,1 ). This leads to the functional relation for the fluorescence intensity with time I(t) = Ap exp − p p+f γt /(p + f ) + A p exp − p p +f γ t /(p + f ), with A and A denoting the amplitudes of the two exponential components.
We fit the measured data in Figs. 5(c, d)

Conclusion and outlook
In conclusion, we demonstrated that by using an oil-immersion objective to image the inner surface of a home-made thin vacuum window, the adsorption, translation and rotational diffusion of single molecules on a surface under UHV can be studied with high spatial and temporal resolutions. The dispersed diffusional dynamics suggest the co-existence of different adsorption states of perylene molecules. This is further supported by the temporal dynamics of the ensemble fluorescence intensity. Uncovering the full complexity of the system would require future work to combine ab initio calculations with complementary measurement techniques. Along this line, the fused silica surface can be encapsulated with thin layers suited for surface science studies. For example, transparent dielectric layers could be incorporated through standard deposition or substrate transfer techniques. It should also be possible to apply conductive layers without significantly impeding the optical transmission. For example, conductive oxides such as indium tin oxide have a transmission of about 80% in the spectral range of 400 nm-600 nm even with 500 nm layer thickness . 50 Gold layer with a thickness of 2 nm still permits optical transmission of 60% in the same wavelength range. 51 Two-dimensional materials, e.g., graphene introduces only about 2% absorption per monolayer. These will make the platform compatible with scanning probe techniques, which could enable direct imaging of the adsorption sites with atomic scale resolutions. 8,9,52 Such a system would also facilitate simultaneous correlative electron and light microscopy. 53,54 Alternatively, implementing gas phase photoionization detection schemes will permit independent determinations of the residence times and scattering velocities of desorbed molecules. 16 Lastly, the bonded window can be attached to a UHV flange through glass-to-metal bonding, to replace the rubber seal. This should permit higher bakeout temperatures and lower vacuum levels down to the 10 −11 mbar range.