Single-molecule studies beyond optical imaging: Multi-parameter single-molecule spectroscopy

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Journal of Photochemistry and Photobiology C: Photochemistry Reviews xxx (2017) xxx–xxx

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Journal of Photochemistry and Photobiology C: Photochemistry Reviews journal homepage: www.elsevier.com/locate/jphotochemrev

Invited Review

Single-molecule studies beyond optical imaging: Multi-parameter single-molecule spectroscopy Martin Vacha ∗ , Dharmendar Kumar Sharma, Shuzo Hirata Department of Materials Science and Engineering, Tokyo Institute of Technology, Ookayama 2-12-1-S8-44, Meguro-ku, Tokyo 152-8552, Japan

a r t i c l e

i n f o

Article history: Received 8 September 2017 Received in revised form 10 November 2017 Accepted 30 November 2017 Available online xxx Keywords: Single-molecule spectroscopy Fluorescence Electron microscopy Scanning probe microscopy Atomic-force microscopy Hydrostatic pressure Electric field Magnetic field

a b s t r a c t Single molecule spectroscopy has undergone a remarkable development in the past few decades, and its ability to unmask the unique features of individual molecules has found increasing use in research of soft matter and polymers, in chemistry, as well as in biophysics and biology. In this concise review we bring an overview of the synergy effects that result from combinations of single-molecule and singleparticle fluorescence spectroscopy with other techniques, such as electron microscopy and scanning probe microscopy, or with external-field effects including hydrostatic pressure and electric and magnetic fields. © 2017 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Single-molecule spectroscopy and electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. SMS and TEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. SMS and SEM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Single-molecule spectroscopy and scanning probe microscopies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Correlated SMS and AFM measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Simultaneous (synchronized) SMS and AFM/STM measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 External field effects in single-molecule spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Hydrostatic pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. Effects of electric field and electric charges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.3. Magnetic field effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

∗ Corresponding author. E-mail address: [email protected] (M. Vacha). https://doi.org/10.1016/j.jphotochemrev.2017.11.003 1389-5567/© 2017 Elsevier B.V. All rights reserved.

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Martin Vacha is a Professor in the Department of Materials Science and Engineering at Tokyo Institute of Technology. He received his education from Charles University in Prague, Czech Republic, where he also obtained his Ph.D. in 1991 for work on low temperature high resolution optical spectroscopy of photosynthetic systems. He has extensive research experience in the fields of hole-burning and single-molecule spectroscopy of organic molecules and molecular complexes gained during stays at academic and government research institutions in Japan. His current main research interests are nanoscale physical properties of organic materials and biomaterials studied by single-molecule techniques. Dharmendar Kumar Sharma completed his Ph.D. from IIT Bombay, India in 2014. His Ph.D. research was focused on spatial and dynamic heterogeneities in complex materials and biological systems using single-molecule diffusion dynamics and spectroscopy. At present, he is a postdoctoral researcher at the Department of Materials Science and Engineering, Tokyo Institute of Technology, where is working on exploration of structuralphotophysical relations and luminescence properties of organic/inorganic semiconductor nano-materials using single particle photo/electro-luminescence spectroscopy. Shuzo Hirata received his Ph.D. in applied chemistry from Tokyo University of Agriculture and Technology in 2009. After research experience in the field of organic opto-electronics, he became an Assistant Professor in the Department of Materials Science and Engineering at Tokyo Institute of Technology from 2012. His current research interests include excitonic properties of materials for optoelectronics and photonics applications.

1. Introduction Single molecule spectroscopy (SMS) has undergone a remarkable development from its origins as a technique in lowtemperature high-resolution spectroscopy [1,2] to applications in super-resolution fluorescent microscopy [3]. Its ability to unmask the unique features of individual molecules has found use in lowtemperature physics and optics [4,5], in research of soft matter [6], polymers [7,8], semiconducting nanocrystals [9] and materials [10], in the general field of chemistry [11,12] including catalysis [13] and physical chemistry [14], and increasingly in biophysics and biology [15–17], with a particular emphasis on super-resolution imaging [18–20]. Detection of single molecules allows the measurement of various photophysical parameters related to fluorescence such as spectra and spectral diffusion, fluorescence intensity fluctuations (intermittency or blinking), fluorescence lifetime, excitation and emission polarization, and photon anti-bunching. In the field of, e.g., polymer physics measurement of these phenomena on the level of single chains lead to an unprecedented insight into photophysics of conjugated polymers [21–24]. Apart from the photophysical properties, microscopy of individual molecules enables precise determination of the molecular orientation and location, and using again the example of polymer physics measurement of these parameters reveals nanoscale mechanism of polymer diffusion and relaxation [25–27]. Molecular localization with subdiffraction precision [28] has ultimately lead to the development of the super-resolution fluorescence microscopy [29–31]. Even though the localization-based super-resolution fluorescence microscopy has brought a qualitative improvement in spatial resolution, there is still a considerable gap between the size of many nano-objects and the practically attainable resolution in fluorescence. It is, therefore, challenging to use the same technique and instrumentation for both imaging the size and structure of

a nano-object and for studying its optical properties. A possible solution to this dilemma is a use of synergetic combination of high-resolution microscopy and single molecule spectroscopy. The synergy of the sub-nanometer to atomic resolution in structural imaging and single-molecule sensitivity in photophysical characterization will enable addressing some of the outstanding issues in chemistry, physics or materials science. In the field of chemistry, for example, it can bring the ability to accurately predict the structure of complex synthetic macromolecules or supramolecular constructs and to correlate such structures to physical-chemical properties and location-sensitive reactivity. In solid-sate catalysis it may enable correlation of catalytic activity with the catalyst crystal structure and composition, or with its porous dimensionality and structure. In physics, it will bring together the atomic-level structure of semiconducting nanocrystals, plasmonic nanoparticles, 2D materials and other low-dimensional systems with their optoelectronic properties. In addition, scanning probe techniques will add the possibility of examining or modifying the opto-electronic functionality of such systems and their prototypical devices. As will be shown below, the first steps of many of these envisioned applications have been realized, including works on different types of semiconducting nanocrystals of mainly the II–VI group as well as on the emerging halide perovskites, or on plasmonic nanostructures. One of the aims of this short review is to provide an overview of recent efforts in this direction of research. The Section 2 summarizes technical developments and results obtained by combined transmission electron (TEM) and scanning electron (SEM) microscopies and fluorescence microscopy on individual nano-objects. In Section 3 the combination of single-molecule spectroscopy with scanning probe microscopies is reviewed. Unlike electron microscopy, fluorescence and scanning probe microscopes can be combined within one instrument. This brings an advantage of the possibility of simultaneous imaging with the two techniques, and also of possible manipulation of the nano-object by the probe and its immediate effect on the fluorescence properties. Such manipulations may include mechanical distortions or applications of electric field and current. These types of external effects have been applied on single molecules and nano-objects also by means other than the probes of scanning microscopes, and such works are reviewed in Section 4, with an emphasis on the effects of hydrostatic pressure, electric field and magnetic field on the photophysical properties and processes in single molecules.

2. Single-molecule spectroscopy and electron microscopy 2.1. SMS and TEM Combined imaging by fluorescence microscopy and spectroscopy and by transmission electron microscopy can be done by subsequent measurements of well-marked samples in the two instruments. The biggest challenge is posed by the differences in resolution and view-fields in both methods. Use of very thin silicon nitride plates that are non-fluorescent and transparent for electron beams as substrates for measurements of single semiconductor quantum dots [32] enabled a systematic investigation of the correlation between crystal structure and photophysical properties of individual colloidal quantum dots of CdSe capped with ZnS shell. The results indicated that presence of stacking faults or even polycrystallinity in individual nanoparticles do not cause fluorescence quenching or decrease of quantum yield. Fluorescence polarization studies showed that a 2-dimensional transition dipole resulting in elliptically polarized emission is a generally property of CdSe nanocrystals. Even single-crystal nanoparticles with almost perfect spherical shapes show fluorescence with a significant degree of elliptical polarization (Fig. 1), and the degree appears to increase

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Fig. 1. High-resolution TEM images (index 1), Fourier transform of the images (index 2) and fluorescence polarization ellipsoids (index 3) of four individual CdSe/ZnS core/shell nanocrystals (a–d). Reprinted with permission from Ref. [33]. Copyright (2003) American Chemical Society.

with the presence of lattice imperfections [33]. More recently, the question of correlation between atomic-level crystal structure and photoluminescence properties of the same CdSe/ZnS quantum dots has been revisited using high angle annular dark field (HAADF) electron microscopy (also known as atomic number contrast scanning transmission electron microscopy, Z-STEM) [34]. The Z-STEM revealed a large heterogeneity in the length of individual quantum dots which reflects different amount of shell material in one crystallographic direction. In terms of photoluminescence properties, the quantum dots were classified with respect to fluorescence intermittency (blinking) into fractions with high ratio of ON-times and low ratio of ON-times, respectively. The correlation with the Z-STEM data showed that the low ratio of the ON-times can be caused by stacking faults in the CdSe core or by surface defects due to irregular or imperfect ZnS shell structure (Fig. 2). Later, this work was extended onto ‘giant’ non-blinking CdSe/CdS quantum dots characterized by very thick CdS shell layer [35]. The experiments revealed a large heterogeneity of the photoluminescence quantum yield among individual quantum dots which is correlated with emission lifetime. However, no relationship was found between the quantum yield and the atomic-level structure of the quantum dots, and charging-discharging processes were proposed as the origin of the quantum yield variations. More recently, similar combined TEM and photoluminescence (PL) imaging and characterization was reported on single organometal halide perovskite nanocrystals. Correlation between the size of individual MAPbBr3 nanocrystals and their PL spectral

peaks enabled an estimation of effective carrier mass. Further, it was found that the nanocrystals possess a small number (1–4) of charge trapping sites, and that one such trapping site is dominant in the PL quenching or blinking [36]. Apart from correlating structure and properties of individual nanoparticles, the combination of TEM imaging and singlemolecule fluorescence microscopy can be used to explore nanoscale structure of porous materials and its relation to molecular diffusion properties [37]. TEM images of mesoporous silica film overlaid with traces obtained from tracking of single diffusing dye molecules revealed that the molecular diffusion pathways are correlated with the pore orientation of the two-dimensional hexagonal structure of the film (Fig. 3). The specific structural features of the mesopores strongly influence the dye diffusion behaviour, such as travelling through linear or strongly curved sections of pores, changing speed in the channel structure, and bouncing off of domain walls [38]. 2.2. SMS and SEM Compared with TEM, scanning electron microscopy poses less severe restrictions on the sample substrates and is thus more easily combined with other microscopic techniques including optical. This fact was made use of in the study of photophysical properties of heterostructured semiconducting nanocrystals. Nanocrystals composed of CdSe cores surrounded by rods of a higher bandgap semiconductor, CdS [39], with resulting structures of tetrapods or rods, were studied simultaneously by single-particle PL exci-

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Fig. 2. Typical crystal structure and surface defects observed for quantum dots characterized by low ratio of ON-times in their photoblinking behavior Left: (a) Example of a stacking fault in Z-STEM image; (b) Inverse Fourier transform of the image shows disruption in the wurtzite crystal lattice; (c) Histogram of the ration of ON-times (ON-fraction) for all quantum dots (grey bars) overlaid with quantum dots with stacking faults (blue bars). Right: (a) Example of pinched-shape quantum dot; (b) Example of irregularly-grown shell; (c) Quantum dot structure with lack of shell grown exposing Cd-rich facets; (d) Histogram of the ration of ON-times (ON-fraction) for all quantum dots (grey bars) overlaid with quantum dots with surface defects (green bars). Scale bars are 2 nm. Reprinted with permission from Ref. [34]. Copyright (2015) American Chemical Society.

tation spectroscopy and SEM, and these measurements made it possible to assign two distinct spectral forms observed in the individual nanocrystals to different physical shapes of the rods. An asymmetrical type of nanorod showing ‘bulb’-like structure leads to distribution of the confinement effect due to continuous variation in the rod diameter and to smearing of the excitonic transition which is reflected in broad structureless spectrum.

SEM imaging also enabled a precise determination of position of individual CdSe/Cd core/shell quantum dots placed within gaps of plasmonic bar antennas. Correlation of the position with lifetime and photon-bunching experiments showed that the plasmonic field does not affect the Auger recombination process and leads to enhancement of biexciton emission [40].

Fig. 3. Merging of the single-molecule trajectories and the TEM micrographs of a mesoporous silica film. a) The merged TEM map is redivided into equal-sized squares that are processed by Fourier transform as shown in the insets. The direction of the channels is perpendicular to a straight line through the maxima. The thickness of the black directors, pointing along the pore direction, correlates with the structural quality. b) Diffusion pathways of single molecules through the pore system (blue trajectories) and positions of the polystyrene beads (red crosses). c) The same area as in b is imaged by TEM, with lines indicating the direction of the channels (directors) and yellow crosses marking the centers of the polystyrene beads. d) Final overlay of trajectories on TEM images; this was obtained by fitting to the best overlay of the polystyrene bead positions. e) Enlargement of a region in d) showing the trajectories running along the channels. Reprinted by permission from Macmillan Publishers Ltd, Ref. [38], copyright (2007).

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Fig. 4. Correlated SEM and super-resolution imaging of Au nanorods by single-molecule fluorescence. a) SEM image of a fluorescent bead. The green circle highlights the outline of the bead. b) Reconstructed super-resolution localization image of the same fluorescent bead. The white circle outlines the shape of the bead. The color bar represents the counts of events in each bin. c) Profile of localization event distribution along the white solid line in b). d) and g) SEM images of individual nanorods. e) and h) Localization images of the rods in d) and g), respectively. The color bars represent the counts of events in each bin. f) and i) Localization event distributions along the white solid lines in e) and h), respectively. The white and green blocks highlight the outline of the nanorods. The red solid lines are fitted Gaussian profiles. Reprinted with permission from Ref. [41], http://pubs.acs.org/doi/abs/10.1021/acsnano.5b07294. Contact American Chemical Society for further permissions related to this figure.

Correlation of precise structural information by SEM with single-molecule fluorescence imaging brings qualitatively new insights into interactions of organic dyes with noble-metal plasmonic nanostructures. Super-resolution fluorescence localization of single crystal violet dyes diffusing in the vicinity of immobilized gold nanorods showed that the most probable apparent location of the emitting dyes is at the center of the nanorods (Fig. 4) even though the strongest enhancement of fluorescence is expected near their tips, i.e., 30 nm away from the center. Finite difference time-domain calculations confirmed that while the molecules may be located at the tips where the strongest near-field enhancement happens, the nanorod functions as a farfield antenna which couples with the molecular emission, and the super-resolution localization visualizes the position of the antenna itself rather than that of the emitter [41]. Later, such correlated SEM and single-molecule super-resolution localization imaging was used to study and optimize the localization accuracy by tuning the spectral separation and distance between the dyes and individual gold nanorods [42]. Also, combination of SEM imaging with the microscopic super-resolution method applied to surface-enhanced Raman scattering (SERS) of single rhodamine molecules adsorbed onto aggregates of silver colloidal nanoparticles revealed with high precision the physical location of the plasmonic hot spots [43]. Such nanoscale hot-spot mapping will have important implications for the design of efficient SERS substrates.

Ultimately, most information would be gained from the synergy of combined SEM and optical microscopy in one experimental setup [44]. Such integrated optical-electron microscope has been constructed [45] and used to study the mechanism of PL blinking in single nanocrystals of methylammonium lead halide perovskites [46]. Super-resolution imaging of the emitting sites correlated with the SEM imaging showed that single crystals of sizes up to several hundreds of nanometers emit and blink as a whole, without changes in the apparent location of the emitting center over the course of the measurement. This fact, together with the observed power-law characteristics of the on and off PL times point to trapping and de-trapping of charges as the probable mechanism responsible for the blinking phenomenon. The work was later extended to still larger polycrystals as well as single-crystal microrods of the same perovskite. The combination of SEM, superresolution localization and super-resolution optical fluctuation imaging enabled detection of emitting regions in the polycrystals (Fig. 5). The blinking was proposed to occur by activation and deactivation of a ‘supertrap’ composed presumably of a donor–acceptor pair [47].

3. Single-molecule spectroscopy and scanning probe microscopies Compared to electron microscopy, combined imaging of single nanoparticles by fluorescence microscopy and scanning probe

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Fig. 5. Correlated SEM and photoluminescence (PL) imaging of methylammonium lead halide perovskites. a) SEM image of two polycrystals; the left one was emissive while the right one was nonemissive. PL localizations are superimposed and the color-coding represents the intensity of the PL emission. b) Time transient of the PL intensity where the four distinct intensity levels are indicated with colored dashed lines. c) SEM image overlaid with SOFI image. d) Schematic of three proposed blinking polycrystal segments together with emission localizations. (d1–4) Schematics showing emission intensity pattern (the same color coding as the PL intensity transient) for different combinations of “on” and “off” states of the individual segments. Each combination of the emitting segments corresponds to an intensity level in a). The white crosses indicate the mean position of the localization clusters. Reprinted with permission from Ref. [47]. Copyright (2017) American Chemical Society.

microscopies such as atomic force microscopy (AFM) is technically much easier and can be achieved in modified optical microscope. In the past years, the synergy of optical and scanning probe microscopies has been used mainly in studies of biological objects such as proteins or DNA [48–50]. The combined atomic force and optical microscopes are typically based on inverted fluorescence microscopes often working in the confocal illumination/detection mode [51–54]. To achieve single-molecule sensitivity in the optical part of the experiment it is necessary to eliminate all additional sources of background that might come with the AFM such as the lasers used to monitor the cantilever deflection. Care must be taken also to select cantilevers with low autofluorecsence [55]. On the other hand, the need for high-numerical aperture objective lenses and thin microscope cover glasses in the optical microscope tend to compromise the AFM resolution by increasing the substrate vibrations and surface roughness. 3.1. Correlated SMS and AFM measurements One way to perform the combined AFM and single-molecule experiments is correlated imaging of the same sample areas, similar to the combined electron and optical microscopy. Such correlated imaging of single CdSe/ZnS quantum dots revealed that while in AFM topography images all the nanocrystals appear similar, in the fluorescence image only a portion of the particles are emitting (bright) and the rest are dark. This finding has direct implica-

tions for determination of luminescence quantum yield in bulk solutions, and there has been indeed a direct correlation between single-particle quantum yield, as well as the fraction of the bright particles on one hand, and the bulk solution quantum yield on the other [56]. Also, AFM imaging of CdSe/CdS/ZnS core/shell quantum dots and their dimers and trimers prepared by repeated precipitation enabled unambiguous identification of the individual aggregated forms, and allowed correlation of the aggregate structure with fluorescence spectra and the underlying electronic coupling [57]. In addition to correlated imaging, the tip of an AFM cantilever can be also used to manipulate nano-objects in the sample. In this way, a single cubic gold nanoparticle was positioned precisely with the aim to control its distance from a single CdSe/ZnS quantum dot. It was shown that with decreasing distance from the Au particle the quantum dots turns from a single-photon emitter to a multi-photon emitter presumably due to an increase of biexciton emission rate, and the process is reversible [58]. For artificial light-harvesting antennas prepared by adsorption and self-assembly of modified bacteriochlorophylls on gold nanorods with the aim to mimic bacterial chlorosomes, the correlated AFM and single-particle fluorescence imaging made it possible to relate the particle size (volume) with the emission intensity and spectroscopic parameters [59]. For fluorescently labeled double-stranded DNA bound to the surface of gold nanorods, correlated super-resolution imaging and AFM showed excellent agreement in terms of the shape and orientation of

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Fig. 6. Atomic force (AFM) and reflection micro-spectroscopy of individual fibrilous nanostructures of pseudoisocyanine J-aggregates in a polymer film. A) AFM image in a tapping mode; B) Reflection image of the same location taken in the peak of the J-band at 572 nm; C) Detail of the AFM image; D) Series of reflection spectra taken from the same area as C); E) Three typical reflectance line shapes observed in D); F) Maximum thickness of the J-aggregate nanostructure (taken from the detailed AFM image) and corresponding relative depth of the exciton-polariton-related mode. G) Absorption spectrum of the J-aggregate/polymer film. Reprinted with permission from Ref. [62]. Copyright (2001) American Chemical Society.

the nanorods, although the size of the nanorods was consistently under-estimated in the super-resolution images [60]. AFM imaging has been also combined and correlated with fluorescence intensity and lifetime imaging on TiO2 nanoparticles labeled with porphyrin dye as electron donors. Combination of the techniques allowed obtaining nanoscale morphology and interfacial electron transfer dynamics, as well as location of each porphyrin molecule on the TiO2 surface with relation to its coupling strength [61]. An earlier study on individual fibrous nanostructures of pseudoisocyanine J aggregates related AFM images with reflection optical microscopy and spectroscopy. The high degree of correlation between the fiber structure (dimensions) and localized reflection spectra confirmed the polariton-like nature of the excitations in these molecular structures due to strong coupling between light and the aggregate excitonic states (Fig. 6), and showed that these structures have a potential as self-assembled optical microcavities [62].

3.2. Simultaneous (synchronized) SMS and AFM/STM measurements Simultaneous and synchronized measurements by AFM and optical microscopy on the same single molecules or single nanoobjects provide a possibility not only to carry out structural imaging with sub-optical resolution, but also to actively manipulate the objects by external stimuli such as mechanical force or electric field. This added dimension in single-molecule studies has a potential to provide deeper insight into structural-functional properties of electronic interactions of individual nano-objects. One direction of research involves mechanical manipulation or modification of the single molecules or particles by the AFM tip and examination of the resulting changes in optical properties. Biopolymers such as protein molecules have been studied before using AFM force spectroscopy. In that method, single protein chains are attached to a substrate on one end and to the AFM tip on the other, and controllably stretched to reveal processes such as pro-

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Fig. 7. Simultaneous AFM and fluorescence microscopy of single MEH-PPV nanoparticles. a) AFM force curve measured on a single nanoparticle. The different regimes are schematically shown above the figure; b) Fluorescence intensity trace measured simultaneously with the force curve in a). The dashed line corresponds to background value of fluorescence. c) Ratio Ired /Iblue of the blue (Iblue ) and red (Ired ) components of the fluorescence spectrum, measured simultaneously with the force curve in a). d) Scheme of the proposed model. Reprinted with permission from Ref. [67]. Copyright (2013) American Chemical Society.

tein folding [63]. Combination of such method with fluorescence microscopy has been demonstrated on proteins labeled with a single pair of dyes designed for efficient Foerster resonant energy transfer (FRET). Monitoring the efficiency of FRET upon stretching allows very precise detection of structural changes of the protein chain, and conformational changes on kinase enzyme HPPK have been observed by this method [64]. In a similar approach, protein unfolding has been studied by AFM stretching and simultaneous monitoring of fluorescence intensity from a luminescent AFM tip excited by evanescent wave of total internal reflection illumination mode, leading to resolution of 20 nm steps associated with the unfolding process [65]. Apart from force spectroscopy by stretching, AFM can be used to locally apply pressure on individual molecules or particles. For green fluorescence protein with a characteristic ␤-barrel structure it has been shown that structural deformation leads to fluorescence quenching, and that the quenching is qualitatively different for compression and extension of the protein, probably due to conformational changes of different parts of the barrel structure [66]. A similar approach of mechanical deformation by an AFM tip performed on single nanoparticles of the conjugated polymer MEHPPV resulted in unexpected enhancement of fluorescence intensity and simultaneous blue shift of fluorescence spectra (Fig. 7). These effects which are reversible upon retraction of the tip were explained as resulting from conformational changes of the polymer chains within the nanoparticles [67]. Analogous experiments carried out on truly single-molecule level using TDI–4PDI (TDI – terrylenediimide, PDI – perylenediimide) multichromophores also showed that compressive stress leads to spectral shifts which can

be both reversible and irreversible, with the latter corresponding to transitions between different conformational states of the complex molecule [68]. Further, similar work on individual quantum dots of CdSe/CdS/ZnS revealed that the external pressure exerted by the AFM tip can reproducibly cause both red and blue spectral shifts, depending on the orientation of the nanocrystals with respect to the external force [69]. The above experiments were carried out simultaneously and, as a result, observation of the synchronized optical response to the external perturbation was possible. In other related works, the external perturbation by AFM compression was carried out separately, and the effects were examined by fluorescence microscopy. In this way, large self-assembled porphyrin rings were structurally modified by removing parts of the ring by the AFM tip, and the structures were observed by confocal microscopy [70]. In a similar approach, nanowires formed by H-aggregated perylene molecules were modified by applying external mechanical force with an AFM tip. As a result, the H-aggregate structure which promotes long-range exciton migration in the aggregate nanowires has been disrupted at the locations of the applied force, leading to exciton quenching and modified fluorescence intensity at such locations [71]. Other than mechanical manipulation, electromagnetic interactions between a molecule or particle and the probe tip have been observed and utilized. In the early days of single-molecule spectroscopy, fluorescence lifetimes of individual molecules measured with near-field scanning optical microscope (NSOM) were observed to vary depending on the NSOM tip position, and this observation was explained due to quenching of the emission by

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Fig. 8. Time traces of photoluminescence (PL) intensity (a–f), photon correlation histograms (g–l) and PL lifetime decay curves (m–r) detected from the same single CdSe/ZnS core/shell quantum dot for different distances from Ag tip of the AFM cantilever (indicated above the PL traces in (a–f)); excitation at 465 nm. Increase of the signal at 0 ns in the photon correlation histograms (i–k) for small quantum dot-Ag tip distances indicates increased relative probability of multiphoton emission. Reprinted with permission from Ref. [77]. Copyright (2016) American Chemical Society.

aluminum film coating of the tip [72,73]. The same effect of modified emission rate, both enhanced and quenched, has later been observed using a gold nanoparticle attached to a tip of a probe in aperture-less NSOM, and was theoretically interpreted using a dipole approximation. A characteristic feature of the imaging is a donut pattern-like image of a single molecule with a dip in the center [74]. The same pattern with a dip in the center is observable in fluorescence images of single molecules measured simultaneously by (far-field) confocal microscopy and AFM, due to quenching of the molecular fluorescence by the conductive AFM tip [75,76]. The interaction with a metallic AFM tip has further found application in a study of multiphoton emission from single CdSe/ZnS quantum dots. With decreasing distance between the quantum dot and the silver-coated AFM tip the character of the quantum dot emission changes from single-photon to multiphoton (Fig. 8), and this effect was explained as due to quenching of single-exciton state by energy transfer to the tip [77]. Interactions of nano-objects with electric field/current mediated by a probe microscope have been also induced actively using scanning tunneling microscopy (STM). Biased STM tips have been used to generate electroluminescence (EL) on nanometer scales in films of poly(phenylene vinylene) derivatives deposited on gold films [78–80]. STM-induced EL signal shows intensity fluctuations within surface domains as small as a few nanometers, and these domains correlate with similar regions appearing in topographic imaging. Ultimate demonstration of STM probe-induced EL on truly single-molecule level was reported on a single polythiophene wire

suspended between the STM tip and a gold surface (Fig. 9). Under positive voltage, the EL spectral and voltage dependencies are consistent with fluorescence, and the transitions are influenced by the polythiophene chain conformation and by plasmon modes at the STM junction [81]. 4. External field effects in single-molecule spectroscopy There have been numerous attempts to study external field effects on the optical response of single molecules. In these works the external fields, such as electric and magnetic fields or hydrostatic pressure, have been applied ‘macroscopically’, that is, over the whole sample. It is instructive to compare some of these works to the similar effects observed on the nanoscale by the scanning probe perturbations. In addition, many of these ‘macroscopic’ single-molecule works may provide a perspective and point towards a future direction in the nanoscale research. 4.1. Hydrostatic pressure Application of external hydrostatic pressure is the ‘macroscopic’ equivalent of the compression experiments carried out by AFM tip in the simultaneous SMS and AFM imaging. External pressure has been applied in the early days of single-molecule spectroscopy on pentacene [82] and terrylene [83,84] in low-temperature crystalline matrices. In all cases, the narrow zero-phonon absorption lines showed red spectral shift which grew linearly with increasing

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Fig. 9. a) Scanning tunneling microscope (STM) image of a polythiophene wire polymerized on an Au(111) surface; b) Normalized conductance G/G0 as a function of tipsurface distance for a polythiophene wire suspended in the junction between the STM tip and the surface; c) Conductance dI/dV spectra measured at different tip-surface distances and inverse decay length ␤ as a function of voltage V for an individual wire. d) Light emission efficiency as a function of V; e) Schematic view of a fluorescent polythiophene junction. Reprinted with permission from Ref. [81]. Copyright (2014) by the American Physical Society.

pressure and was reversible. The linear relationship reflects the fact that the pressure-induced shift is a kind solvent shift determined by the attractive part of Lennard-Jones interaction potential [83]. The slopes of the linear relationship differed for different molecules and were more dispersed for the Shpolski matrix of hexadecane [84] compared to crystalline p-terphenyl [82,83]. More recently, acoustic pressure and strain were measured by single molecules of dibenzoterrylene in anthracene crystal mounted on a tuning fork. Fast spectral shifts occurring with the acoustic frequency were detected as broadening of the zero-phonon absorption lines at cryogenic temperatures [85]. Room-temperature experiments under applied external pressure have proven to be more difficult because of the need of high numerical aperture objective lens. A pressure cell capable of withstanding pressures of 1.2 kbar which can be mounted on a fluorescence microscope has been demonstrated by observing fluorescence from single Alexa molecules [86]. An alternative design for lower numerical aperture objective lenses for pressures up to 1.4 kbar was used to study the effect of external pressure on the rotation of F1-ATPase. The pressure did not affect the rotation steps or directionality but revealed the existence two pressure-sensitive reactions, one of which was an ATP binding [87].

4.2. Effects of electric field and electric charges There has been a strong record in research on the effect of electric field and/or electric current on the emission of single molecules, and many of the concepts might be potentially developed further

by combination with probe microscopies. The effect of externally applied electric field on the energy of an optical transition which is demonstrated as spectral shift is known as Stark effect, and has been explored extensively from the early days of single-molecule spectroscopy. The facts that the ensemble averaging is removed and that at cryogenic temperatures the extremely narrow linewidth of single molecules is sensitive to smallest perturbations make single-molecule excitation spectra of rigid aromatic dyes an excellent tool to distinguish linear and quadratic Stark effects [88–90], explore higher-order [90,91] and anomalous [92] effects in crystals and Shpolskii matrices, and reveal the inhomogeneous nature of polymer films [93]. Later, linear Stark effect with spectral shifts on the order of nanometers and exhibiting hysteresis loops has been observed for the conjugated polymer MEH-PPV at cryogenic temperatures [94]. A combination of linear and quadratic Stark effect was observed also at cryogenic temperatures for single quantum dots of CdSe, pointing to the presence of both environmentallyinduced permanent dipole and high polarizibility of the excited state [95]. The enhanced response to the electric field effects in quantum dots compared to organic dyes is a result of quantum confinement and known as quantum-confined Stark effect. Later, the quantum-confined Stark effect was used in the study of electric field effect on emission of single quantum rods (QR) of the same semiconductor CdSe [96]. External field applied on individual QRs caused reversible switching (both OFF and ON) of the emission, which was preceded by continuous non-linear red spectral shift. In addition, discrete spectral jumps observed in some of the QRs were attributed to charged exciton emission.

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Fig. 10. Spectral trails of zero-phonon excitation lines of terrylene derivative molecules deposited on germanium-doped indium-tin oxide (ITGO) as a function of applied dc voltage. In the top trails, most molecules shift to the red, the bottom trail shows a molecules with a blue shift. The experiments were done at cryogenic temperatures (1.8 K). Reprinted with permission from Ref. [97]. Copyright (2001) by the American Physical Society.

Stark effect and related phenomena are also responsible for nanoscale effects monitored by single-molecule emission in charge and/or current carrying systems. At cryogenic temperatures, single molecules of terrylene derivatives deposited on top of ZnO or doped ITO semiconductors respond strongly to dc and ac currents in the substrate by spectral shifts and resonances (Fig. 10). These results have been explained by changes in polarizability in addition to the Stark shift [97]. In a similar experiment, chargetransport phenomena were studied in a field-effect transistor fabricated from terrylene-doped anthracene crystal. Large spectral shifts of the single terrylene molecules due to localized Stark effect reflect the charge dynamics and distributions of charge traps [98]. Charges also strongly influence emission of single molecules in organic electroluminescence devices (OLED) where fluctuations

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of the charge pathways cause strong stochastic blinking of EL of small molecules of organometallic iridium complexes [99] as well as conjugated polymers of polyfluorenes [100]. In the latter, large spectral shifts occurring during the OLED device operation have also been observed, and even though these have been explained due to charge-assisted aggregation effects, contribution from localized Stark effect may also be playing a role. Very recently, the effects of charges have been observed as spectral shifts of single perrylene bisimide molecules in polymer matrices upon photobleaching even without external electric field applied [101]. Other effects that externally applied electric field can cause in single molecules are luminescence quenching and blinking. The effect of quenching has been studied for conjugated polymers, and it was argued that intensity fluctuations and quenching of single clusters [102] or single chains [103] of MEH-PPV were caused by the field-induced generation of quenchers such as polarons. A controlled injection and retraction of charges on single molecules of the same conjugated polymer using a purposely built device later indicated that the blinking and bleaching mechanism in the PPV family of conjugated polymers (Fig. 11) is likely caused by reversible photooxidation [104]. Similar approach has been used for single nanoparticles of polyfluorene where it was shown that controlled and reversible hole injection into the nanoparticles leads to population of deep hole traps which cause quenching of the fluorescence [105]. In the case of single chains of polythiophenes, on the other hand, it was argued that field-induced quenching could be caused by direct exciton dissociation due to lower exciton-binding energy in this class of conjugated polymers [106]. Effect of external electric field on single molecule blinking has been observed on single molecules of ATTO dye dispersed in the conductive matrix of polyvinylcarbazole. Fluorescence blinking in the system is generally caused by forward and backward electron transfer from the dye to the matrix, and these processes can be both enhanced and inhibited depending on the orientation of the electron donating and accepting sites with respect to the applied field, leading to the possibility of external control of the blinking [107]. In a non-conducting matrix of PMMA, electric field-induced quenching or alternately enhancement of fluorescence of squaraine derivatives was explained by diversity in field-induced polarization of the matrix [108]. For an organic dye SAMSA covalently bound to Au nanoparticles it was found that single molecules respond strongly to external electric field by changes in blinking dynamics and that the optical response has a resonance behavior with characteristic frequencies on the order of few Hz [109]. Finally, in core–shell CdSe/CdS nanocrystals the blinking mechanism has long been attributed to charging of the particles. Control of the degree of charging of individual nanocrystals directly immersed in electrolyte in an electrochemical cell revealed two types of blinking, only one of which is correlated with changes in PL lifetimes [110]. The two types were assigned to reversible charging of the nanoparticle core and to charge fluctuations on its surface, respectively. An interesting concept of electrically controlled energy transfer has also been demonstrated. Förster resonant energy transfer requires proximity of the donor and acceptor molecules and overlap of their emission and absorption spectra. External electric field can be used to engineer the overlap and this has been realized for donor-acceptor pairs of CdSe/CdS nanorods and Cy5 dyes. For pairs that do not show any overlap, external electric field can cause red shift of the donor spectrum via quantum-confined Stark effect (Fig. 12), thus turning on the FRET process [111]. In a similar approach, FRET from single quantum dots of PbS/CdS to graphene has been controlled electrically by tuning the spectral overlap by controlling the graphene optical transitions [112].

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Fig. 11. Top: Configuration of the single-molecule charging/discharging device. The fluorescence trace was measured from a single MEH-PPV conjugated polymer chain at zero applied bias voltage. The trace shows a prolonged OFF-time due to blinking. Bottom: Fluorescence trace of another single MEH-PPV chain periodically modulated by the applied triangular bias ±10 V. An OFF-period of a similar duration as in the top panel shows recovery of fluorescence which is modulated by the bias, pointing to photooxidation as a mechanism for the blinking. Reprinted with permission from Ref. [104]. Copyright (2004) American Chemical Society.

4.3. Magnetic field effects Compared to the external electric field, the effect of magnetic field on the spectra of single molecules has been studied to much less extent. The counterpart of Stark effect is Zeeman effect, which has been observed on single pentacene [113] and terrylene [114] molecules. External magnetic field leads to spectral shift which is observable on zero-phonon absorption (excitation) lines at cryogenic temperatures. For pentacene in para-terphenyl crystal, changing the direction of the magnetic field enabled a determination of molecular orientation in two distinct sites in the matrix [113]. Terrylene in a Shpolskii matrix of hexadecane exhibited quadratic red shift, which was explained due to paramagnetic contribution [114]. More recently, external magnetic field has been used to control fine-structured energy levels of CdSe quantum dots at cryogenic temperatures [115,116]. In materials specifically designed to stabilize an excited state of a singly-charged nanocrystal (the so-called trion state), a CdSe/CdS4 /ZnS1 core/shell/shell structure was prepared so that the large core minimizes Auger effects, the CdS shell enables electron access to the nanocrystal surface and a thin outer ZnS shell provides chemical stability. Such quantum dots show stable emission from the charged state (trion emission) at cryogenic temperatures and enable detection of the line-splitting in external magnetic field. These magneto-optical studies provided the electron and hole g-factors and verified the trion charge. Increasing splitting with increasing magnetic field also nicely demonstrated the onset of spin relaxation at energies cor-

responding to the lowest acoustic phonons, thus illustrating the acoustic phonon bottleneck effect [115]. Fine structure of bandedge energy levels in the CdSe/CdS4 /ZnS1 quantum dots can be also induced by structural asymmetry of the CdSe particle core which lifts a degeneracy of the states. Such fine-line structure is measurable at cryogenic temperatures and enables the study of the Zeeman effect. Depending on the orientation of the external magnetic field with respect to the anisotropic nanocrystal ellipsoidal axis the Zeeman effect can cause both increase and decrease of the line splitting with increasing magnetic field [116]. While not strictly an external magnetic field effect, a related phenomenon of magnetic resonance of a single spin of a single molecule has been studied by optical means [117,118]. The experiment of optically-detected magnetic resonance (ODMR) involves monitoring fluorescence from a single molecule upon irradiation with frequency scanned microwave radiation. At the microwave frequencies which match the zero-field splitting of the molecular triplet state a redistribution of the population occurs, resulting in a drop of fluorescence intensity. This technique has later been used extensively for the study of photoluminescence from nitrogenvacancy (NV) centers in diamond nanoparticles on the level of single defects [119]. Unlike organic dyes, NV center optical transitions involve triplet ground electronic state which enables efficient spin manipulation. This feature has been used, e.g., for nanoscale magnetic sensing [120] or for nanoscale detection of nuclear magnetic resonance [121].

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Fig. 12. Electrical tuning of resonant energy transfer from nanocrystals to single carbocyanine dye molecules using the quantum-confined Stark effect at 50 K a) Dependence of the PL spectrum of a single nanocrystal on electric field in the absence of dye molecules; b), c) Modulation of the fluorescence of two different nanocrystal–dye couples by cyclic application of a bias. Depending on the spectral position of the nanocrystal, Foerster resonant energy transfer occurs either with b) or without c) an electric field, so that the dye emission switches on and off with a change in field. Reprinted by permission from Macmillan Publishers Ltd, Ref. [111], copyright (2006).

5. Conclusions We have presented a concise review of the combinations of single-molecule and single-particle optical spectroscopy with electron and scanning probe microscopies and external field effects. The article which is meant as a brief overview of what has been done using these techniques, introduces the many highly innovative ideas on the possible synergies resulting from these combinations, and will hopefully inspire further research in this direction. The chosen focus of the review on fluorescence from single molecules or nanoparticles meant that many closely related techniques have not been included in the overview. Near-field scanning optical microscopy, aperture-less tip-enhanced microscopy or plasmonics are such examples, and reviews of these methods can be found elsewhere [122–125]. Also, spectroscopy of single molecules spatially confined in anti-Brownian electrokinetic traps is related to the topic of this review in the sense that it uses electric field as the trap driving force, and readers are referred to an overview of this technique [126]. Similarly, the broad field of single-molecule manipulation using optical tweezers and related techniques is not included and has been reviewed elsewhere [127].

As pointed out in the introduction, the synergetic combinations of sub-nanometer or atomic resolution in structural imaging and single-molecule sensitivity in photophysical characterization have a potential to address many of the outstanding issues in chemistry, physics or materials science. Examples of some of such applications have been presented in this review. More widespread use of the combined electron and optical microscopy could be aided by commercial availability of an integrated microscope, such as the one introduced in Ref. [45]. In comparison, the combinations of fluorescence and scanning probe microscopies are technically less demanding and offer the added dimension of the active manipulation/control of various phenomena. The possibility to locally apply external pressure could be used, for example, in nanoscale characterization of the mechanochromic effect in such class of materials. Also, the whole field of force spectroscopy that has been developed for polymers and biopolymers could be potentially extended to the study of excitonic coupling or aggregation effects on conjugated polymers, supramolecular structures, molecular aggregates, etc. The potential to use the scanning probes as localized sources of electric or magnetic fields has been essentially unexplored and could offer promising possibilities both in the studies of basic phe-

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