We report on a thermal imaging technique based on fluorescence polarization anisotropy measurements, which enables mapping the local temperature near nanometer-sized heat sources with 300 nm spatial resolution and a typical accuracy of 0.1 degrees C. The principle is demonstrated by mapping the temperature landscape around plasmonic nano-structures heated by near-infrared light. By assessing directly the molecules' Brownian dynamics, it is shown that fluorescence polarization anisotropy is a robust and reliable method which overcomes the limitations of previous thermal imaging techniques. It opens new perspectives in medicine, nanoelectronics and nanofluidics where a control of temperature of a few degrees at the nanoscale is required.

1 Molecular Nano-Optics and Spins (MoNOS), Leiden Institute of Physics, Leiden University 2 Molecular Cell Biology, Institute of Biology, Leiden University 3 Department of Biophysics, Leiden Institute of Physics, Leiden University * current address: Centro de Química Estrutural – Complexo I, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal † current address: ICFO – Institut de Ciències Fotòniques, Mediterranean Technology Park, 08860 Castelldefels (Barcelona), Spain

We demonstrate a novel technique to achieve fast thermal cycles of a small sample (a few femtoliters). Modulating a continuous near-infrared laser focused on a metal film, we can drive the local temperature from 130 to 300 K and back, within a few microseconds. By fluorescence microscopy of dyes in a thin glycerol film, we record images of the hot spot, calibrate its temperature, and follow its variations in real time. The temperature dependence of fluorescence anisotropy, due to photophysics and rotational diffusion, gives a steady-state temperature calibration between 200 and 350 K. From 200 to 220 K, we monitor temperature more accurately by fluorescence autocorrelation, a probe for rotational diffusion. Time-resolved measurements of fluorescence anisotropy give heating and cooling times of a few microseconds, short enough to supercool pure water. We designed our method to repeatedly cycle a single (bio)molecule between ambient and cryostat temperatures with microsecond time resolution. Successive measurements of a structurally relevant variable will decompose a dynamical process into structural snapshots. Such temperature-cycle experiments, which combine a high time resolution with long observation times, can thus be expected to yield new insights into complex processes such as protein folding.

Single nano-objects display strong fluctuations of their fluorescence signals. These random and irreproducible variations must be subject to statistical analysis to provide microscopic information. We review the main evaluation methods used so far by experimentalists in the field of single-molecule spectroscopy: time traces, correlation functions, distributions of "on" and "off" times, higher-order correlations. We compare their advantages and weaknesses from a theoretical point of view, illustrating our main conclusions with simple numerical simulations. We then review experiments on different types of single nano-objects, the phenomena which are observed and the statistical analyses applied to them.

An artificial light-harvesting dendrimer showing highly efficient electronic excitation energy transfer from four peripheral donors to one central acceptor has been investigated by single-molecule spectroscopy at low temperatures. Confocal imaging in combination with frequency selective excitation spectroscopy gives direct access to energy transfer rates of individual donors and allows the determination of energy transfer pathways within a single multichromophoric aggregate.

Photobleaching is a severely limiting factor in the optical study of single biomolecules. We investigate the photobleaching of rhodamine 6G (R6G) ensembles in poly(vinyl alcohol) (PVA) as a function of illumination time, excitation intensity, the presence of oxygen, and temperature. We observe nonexponential kinetics related to primary photobleaching through two dark states-the triplet state and a radical anion-and to secondary photobleaching after the optical excitation of those dark states. Reactions of the metastable states with oxygen can lead either to photoproducts or to a recovery of the ground state. Oxygen can therefore enhance or reduce photobleaching, depending on the experimental conditions. At low temperature, photobleaching is reduced although not completely suppressed. Despite the presence of the long-lived radical anion, we are able to observe single R6G molecules in PVA. At room temperature, only relatively bleaching-resistant molecules are resolved as individuals. At low temperature, the observation times become considerably longer. Our study shows that metastable states other than the triplet drastically affect photobleaching.

Publisher Summary This chapter discusses the single molecule spectroscopy of multichromophoric dendrimers at room and liquid helium temperatures. Single molecule spectroscopy has become an important tool for the optical study of the electronic interactions of proximate chromophores as the electronic interactions can be studied without averaging over the properties of an intrinsically heterogeneous ensemble. Single perylenemonoimide (PMI) chromophores are selectively addressed in the frequency domain within spatially isolated multichromophoric dendrimers, which are dispersed in a polymer film. Although the chromophores are interacting, they still can be addressed separately. This feature in connection with the high spectral resolution at low temperatures opens up a number of promising perspectives for the study of proximate and interacting molecules. By combining confocal microscopy and high-resolution laser spectroscopy at low temperatures, single chromophores have been spectrally isolated within two chemically distinct multichromophoric dendrimers dispersed and spatially isolated in a polymer film. It is shown that unidirectional Forster type energy transfer among the chromophores leads to emission from the chromophoric site, which is lowest in transition energy.

We have conducted single-molecule spectroscopy of a fluorescent polyphenylene dendrimer consisting of four peripheral perylenemonoimides which serve as energy donors and a central terrylenediimide which is the energy acceptor. After selective excitation of the donors the low-temperature emission spectra of single dendrimers show the purely electronic zero-phonon line as the most prominent feature of the acceptor. These sharp emission lines are subjected to appreciable spectral shifts. Fluorescence excitation spectroscopy of individual dendrimers in the spectral region of the donor absorption allows to extract energy transfer rates for single donors within the dendrimer. Although the energy transfer from perylenemonoimide to terrylenediimide is quite efficient, energy transfer between two proximate perylenemonoimides might be a competing process. This is shown by experiments with molecular dimers, in which two perylenemonoimides are held at a short distance by a benzil spacer.

The conformational dynamics of proteins, a key issue in molecular biology, are characterized by the existence of a complex energy landscape, leading to a potentially huge number of possible folding routes for a given protein. This presents the experimenter with the challenge of following simultaneous rapid transitions between many different conformational states. Fluorescent labeling allows one to conduct optical experiments on individual protein molecules which naturally avoids the difficulties associated with unsynchronized ensembles. However, single-molecule experiments on biomolecules at room temperature only give access to their dynamics on a limited timescale

We have investigated multichromophoric assemblies composed of a variable number of chromophores by single molecule spectroscopy. Specific spectral signatures have allowed to distinguish at the single molecule level between molecular dimers representing strong and weak electronic dipole-dipole coupling. By using fluorescence excitation spectroscopy at low temperature, the sharp purely electronic zero-phonon lines of single chromophores within a single dendrimer containing four chromophores could be isolated in the frequency domain.

Isolated multichromophoric dendrimers containing four perylenemonoimide chromophores were investigated in a confocal microscope which allowed imaging of the same set of dendrimers over a wide temperature range. Emission spectra at low temperature (2 K) reveal narrow zero-phonon lines although the electron-phonon coupling is not negligible. By using fluorescence excitation spectroscopy, the sharp purely electronic zero-phonon lines of single chromophores within a single multichromophoric unit could be isolated in the frequency domain. Selective excitation into a higher energy chromophoric site is followed by rapid directional energy transfer into the lowest energy site from which emission then occurs.

We investigate the fluorescence intensity of rhodamine 6G in poly(vinyl alcohol) as a function of excitation intensity, illumination time, the presence of oxygen, and temperature. The variations in emissivity (or fluorescence brightness) are attributed to a dark state, which shows populating kinetics resembling those of the triplet state, but a much longer lifetime. We simulate the observed kinetics by a four-level model, in which a long-lived dark state is formed through the triplet as an intermediate state. The weak temperature dependence of the lifetime of the dark state points to electron tunneling as the main recovery process. This intermolecular mechanism also explains the observed broad distribution of lifetimes. An electron-spin-resonance experiment confirms the assignment of the dark state to a radical. For the first time, photoinduced charge transfer is identified as a source of blinking in single-molecule measurements.

Photobleaching and photoblinking have proven to be the main bottleneck for single-molecule microscopy and spectroscopy at room temperature. Here, a quantitative ensemble study of the kinetics of photoblinking and photobleaching at room temperature of a typical fluorescent label, Rhodamine 6G, in a polar, hydrogen bonding, solid matrix of polyvinylalcohol is presented as a function of the excitation intensity and the presence of oxygen. To achieve uniform irradiation of all molecules present in the excitation focus, the sample (2.0 x 10–5 M R6G in PVA spin-coated on a quartz substrate) is covered by a pinhole array mask with holes of diameter 40 ∝m, each addressable as an individual sample. The experiments are performed at intensities between 65 mW/cm2 and 320 W/cm2 and the measured emissivity of the system is normalized to that at 65 mW/cm2. The emissivity is shown to decrease by a factor of up to 20 at high intensity indicating the presence of a dark state, which would lead to photoblinking of single molecules. The triplet state of R6G cannot be this dark state, as it is hardly populated at excitation intensities below 1 kW/cm2. However, our data suggest that this state might be an intermediate between the singlet excited state and the dark state, which could be for instance a radical. Fig. 1 shows long-term fluorescence traces obtained at room temperature in an air atmosphere, displaying photobleaching. The rates governing blinking and bleaching are found to be widely distributed. Bleaching is shown to be more efficient in air, while blinking is more pronounced in the nitrogen atmosphere, because the lifetime of the dark state becomes longer.

The photooxidation of single dye molecules can be followed by confocal fluorescence microscopy. The self-sensitized reaction with singlet oxygen leads to a suite of products, which may be differentiated spectrally. Tentative structures for certain photoproducts have been obtained from quantum-chemical calculations.

Persistent spectral hole-burning of dopant chromophores embedded in solid matrices has proven to be a sensitive high-resolution spectroscopic tool to investigate structural and dynamic properties of amorphous and crystalline hosts at low temperature []. A commonly encountered mechanism of holeformation is the nonphotochemical process, for which it is assumed that the frequency selective laser excitation and the subsequent relaxation of guest and host eventually leads to a change of configurational degrees of freedom in the nearby environment of the photo-excited centers or in the impurities themselves (or both) []. However, detailed knowledge about the microscopic mechanism of the nonphotochemical process is rare. Methyl group spin conversion [] and rearrangement of hydrogen bond networks [] belong to the few mechanisms known in the literature. In the present work we want to introduce a model system which allows the reproducible observation of nonphotochemical hole-burning at the single molecule level, a phenomenon which amounts to the controlled optical manipulation of an isolated chromophore. We will illustrate how a number of experimental techniques available in single molecule spectroscopy can be combined to obtain ample information about the underlying hole-burning mechanism. Then we will introduce a theoretical approach [] to elucidate the microscopic nature of the configurational degrees of freedom responsible for the formation of the photoproduct: the results of recent molecular dynamics simulations do indeed admit a detailed mechanistic scenario for the hole-burning process in our model system. We thus hope to demonstrate how studies at the single molecule level can serve to improve our understanding of the structural dynamics of solids at low temperatures. To do so, we will start by giving a brief overview of the basic concepts of low-temperature single-molecule spectroscopy.

By applying an external electric field, we have studied the de Stark shifts at the two states of a low-temperature single-molecule optical switch. We observe a reversible change from a mainly quadratic field dependence to a linear field dependence. The data analysis provides the S-1 -S-0 dipole moment and polarizability differences of both the original and photoproduct state. On the basis of these data, a detailed microscopic scenario for the underlying nonphotochemical hole burning mechanism is discussed.

This paper reports on fluorescence microscopy and spectroscopy of single terrylene molecules embedded in p-terphenyl at room temperature. The basic photophysical parameters of this crystalline host/guest system such as photostability, single-molecule emission rates and the properties of dispersed fluorescence spectra are reported. The stability of the single-molecule signals allowed the direct observation of singlet–triplet quantum jumps at room temperature, by means of which the underlying intersystem crossing rates can be investigated. The results of earlier investigations at liquid-helium temperatures are reviewed briefly where appropriate. The properties of terrylene in p-terphenyl are also compared to those of other host/guest systems at room temperature.

This paper reports on frequency jumps of single molecule excitation lines: The first part deals with spontaneous spectral jumps (spectral diffusion) and gives examples of what can be learned from investigations of spectral diffusion about the low temperature physics of amorphous solids. The second part of this article discusses light-induced frequency jumps in the crystalline system terrylene in p-terphenyl where reversible single molecule hole-burning allows the optical manipulation of single absorbers in a remarkably controlled and reproducible fashion. Furthermore we demonstrate how this system permits investigations of one and the same chromophore over a period of a few weeks at least.

The controlled manipulation and switching of single atoms and molecules raise the prospect of ultra-high-density data storage. Switching by motion of a single atom has been reported1, and techniques of single-molecule optical detection and spectroscopy2 in the condensed phase have been refined to a degree that allows the modification of the absorption properties of a single chromophore3. Light-induced jumps in single-molecule excitation frequencies have been reported3,4,5, but in none of these cases could the process be controlled: the jumps varied from molecule to molecule, they were interrupted by spontaneous jumps, and the new excitation frequencies could not be identified unambiguously. Here we report light-induced reversible frequency jumps ofsingle molecules of the aromatic hydrocarbon terrylene embedded in a particular site of a p-terphenyl host crystal6 at temperatures of around 2 K. The changes in absorption frequency for different terrylene molecules were identical (within 0.5%) for all samples studied. Thus we were able to switch single-molecule absorption lines in a controlled way between well-defined frequency positions.

This paper reports spectroscopic investigations of the chromophore terrylene embedded in a matrix of crystalline p-terphenyl. While this system is particularly well suited for single molecule spectroscopy, little is known about the guest site configuration of terrylene. To shed some light on this issue, we employed absorption and fluorescence spectroscopy and compared the experimental data to the results of theoretical calculations. Based on this comparison we suggest a substitution scheme which is in agreement with all the spectroscopic evidence. The dispersed fluorescence spectra of single molecules in the wings of the inhomogeneous distribution deviate significantly from the bulk spectra. This observation is discussed in terms of a host-induced change of the structure and a possible C13 isotopic substitution of the chromophores. Finally we investigated the dynamic host–guest interactions via the temperature-dependent shift and broadening of single molecule excitation lines and found these processes dominated by coupling to characteristic pseudolocal phonon modes of the host, although chromophores in the wings of the inhomogeneous distribution exhibit additional contributions which we attribute to thermal matrix expansion.