Stimulated emission depletion following two photon excitation

The technique of stimulated emission depletion of fluorescence (STED) from a two photon excited molecular population is demonstrated in the S, excited state of fluorescein in ethylene glycol and methanol. Two photon excitation (pump) is achieved using the partial output of a regeneratively amplified Ti:Sapphire laser in conjunction with an optical parametric amplifier whose tuneable output provides a synchronous depletion (dump) pulse. Time resolved fluorescence intensity and anisotropy measurements of the fluorescein emission are made using picosecond time-correlated single photon counting. Pump-dump time delayed fluorescence intensity measurements are used to characterise the response of the system and to provide additional data on saturation dynamics of the dump transition. Two photon STED is modelled using both approximate analytical techniques in the weak dump limit and by numerical solutions to the appropriate rate equations. The latter are used to fit experimental data from which it is possible to determine the cross-section for the stimulated transition and lifetime of the upper vibrational levels of the ground state

Single and two photon time resolved polarised fluorescence studies of probe molecule dynamics in nematic liquid crystals

We present the results of combined single and two photon linearly polarised time resolved fluorescence anisotropy measurements of the order and motion of a fluorescent probe (rhodamine 6G) in the nematic phase of 4-n-pentyl4'cyanobiphenyl (5CB). Variation of the excitation polarisation angle (P) with respect to the nernatic director yield a set of initial single and two photon anisotropies R(0,beta). Single photon R(0,beta) measurements yield the [P-2] and [P-4] moments of the ground state orientational distribution function. For rhodamine 6G in 5CB these indicate that the inclusion of higher moments ([P-6] and above) are necessary to describe the probe ordering within the nematic host.. Two photon R(0,beta) measurements however allow the direct measurement Of [P-6], for rhodamine 6G these yield a value close to theoretical predictions. Two and single photon initial anisotropy measurements are wholly consistent with an approximately Gaussian probe distribution at an angle of 38degrees to the nematic director with a full width half maximum of c.a. 26degrees. Variation of beta affords the photoselection of both cylindrically symmetric and asymmetric degrees of probe alignment that are sensitive respectively to theta and theta plus phi diffusion in the laboratory (nematic director) frame. Cylindrically symmetric and asymmetric alignment relaxation are observed to be linear but with distinctly different relaxation rates, indicating highly restricted probe motion within the nernatic environment

Polarised stimulated emission depletion studies of two-photon excited states

Stimulated emission depletion (STED) population and polarisation dynamics following two-photon excitation are investigated for rhodamine 6G in ethylene glycol. Time resolved fluorescence intensity and polarisation measurements were made using picosecond time-correlated single photon counting (TCSPC). Cross-sections for the stimulated transition were measured between 614nm (2.32 X 10(-16) cm(2)) and 663.5nm (6.05 X 10(-17) cm(2)), ground state vibrational lifetimes were found to vary between 314fs and 467fs. A collinear (180degrees) excitation-detection geometry was employed to investigate re-polarisation of the excited state array yielding fluorescence anisotropies above the two-photon limit. The circumvention of single-photon selection rules is demonstrated allowing the measurement of higher order parameters and correlation functions that are wholly inaccessible to 'conventional' (spontaneous) time resolved fluorescence techniques

Time-resolved fluorescence and FCS studies of dye-doped DNA

Fluorescence lifetime, anisotropy and intensity dependent single molecule fluorescence correlation spectroscopy (I-FCS) are used to investigate the mechanism of fluorescence saturation in a free and nucleotide bound fluorophore (NR6104) in an antioxidising ascorbate buffer. Nucleotide attachment does not appreciably affect the fluorescence lifetime of the probe and there is a decrease in the rate of intersystem crossing relative to that of triplet state deactivation. The triplet state fraction is seen to plateau at 72% (G-attached) and 80% (free fluorophore) in agreement with these observations. Measurements of translational diffusion times show no intensity dependence for excitation intensities between 1 and 105kW cm-2 and photobleaching is therefore negligible. The dominant mechanism of fluorescence saturation is thus triplet state formation. I-FCS measurements for Rhodamine 6G in water were compared with those in the ascorbate buffer. In water the triplet fraction was saturated at considerably higher powers (45% at ca. 1.5 × 103kW cm-2) than in the ascorbate buffer (55%ca. 1 1kW cm-2)

Stimulated emission depletion studies of molecular probe dynamics

Stimulated emission depletion (STED) population and polarisation dynamics are used to determine (α40 the degree of hexadecapole alignment created in ensembles of rhodamine 6G molecules in solution following two-photon excitation. Hexadccapolo molecular alignment is an unavoidable consequence of two-photon excitation but is not observed in spontaneous emission. For a single element diagonal transition tensor measurements of the fluorescence anisotropy R(t) in systems undergoing small step isotropic rotational diffusion can in principle be used to determine (a40)- STED measurements of rhodamine 6G yield a value for (α40) tnat is larSer tnan tnat predicted for a single element transition tensor (Sxx). Recent work in our laboratory indicates that whilst Sxx is dominant Syy, Sxy and Syx are finite, measurements of (α40} appear to be a sensitive probe of the structure of the two-photon transition tensor. STED and fluorescence anisotropy measurements are extended to Rhodamine 6G in the isotropic phase of 5CB a system where small step Isotropie rotational relaxation does not take place. Here the values of 〈α40〉 are considerably larger. These results are discussed in terms of the initial hexadecapole alignment and 〈α40〉 relaxation dynamics in a restricted geometry

Stimulated emission depletion and fluorescence correlation spectroscopy of a branched quadrupolar chromophore

Stimulated emission depletion (STED) and single molecule fluorescence correlation spectroscopy (FCS) are used to determine stimulated emission cross-sections and investigate non-radiative relaxation in a branched quadrupolar chromophore (OM77). The results are used as inputs to simulations of single molecule STED by which the feasibility of STED control of the single molecule fluorescence cycle call be assessed. Single molecule STED in OM77 is shown to be readily achievable; however its effectiveness in reducing triplet trapping is apparently mediated by fast non-radiative relaxation processes other than intersystem crossing and rapid quenching of the triplet state in a non-deoxygenated environment

Stimulated emission depletion dynamics in push-push polyenes

Two-photon fluorescence polarisation and stimulated emission depletion dynamics are investigated in three high two -photon cross-section push-push polyenes: OM62, LP79 and OM77 and compared to the behaviour of a standard fluorophore (rhodamine 6G). Two-photon fluorescence anisotropy measurements (R(O) and Omega) were undertaken using picosecond time-correlated single photon counting (TCSPC). For OM62 and LP79 these are consistent with a diagonal two-dimensional transition tensor with S-XX>S-YY. For OM77 the contribution of off-diagonal elements (S-XY & S-YX) appears significant. Two-photon fluorescence anisotropy decay data is combined with streak camera measurements of excited state population depletion to determine stimulated emission cross sections and ground state vibrational relaxation times. Cross-sections for STED in all three polyenes were found to be significantly higher than those for rhodamine 6G. The efficiency of STED is however dependent on the degree to which the S-1-->S-0 transition is saturated by the DUMP pulse; this is mediated by fast ground state vibrational relaxation. Of the three polyenes.. LP79 is seen to combine a large stimulated emission cross-section (c.a. 13sigma(r6G)) with rapid ground state relaxation (tau(R)=240fs)

Developments in the Photonic Theory of Fluorescence

Conventional fluorescence commonly arises when excited molecules relax to their ground electronic state, and most of the surplus energy dissipates in the form of photon emission. The consolidation and full development of theory based on this concept has paved the way for the discovery of several mechanistic variants that can come into play with the involvement of laser input – most notably the phenomenon of multiphoton-induced fluorescence. However, other effects can become apparent when off-resonant laser input is applied during the lifetime of the initial excited state. Examples include a recently identified scheme for laser-controlled fluorescence. Other systems of interest are those in which fluorescence is emitted from a set of two or more coupled nanoemitters. This chapter develops a quantum theoretical outlook to identify and describe these processes, leading to a discussion of potential applications ranging from all-optical switching to the generation of optical vortices

Cidades Espraiadas: porque o modelo americano deve ser evitado no Brasil

We present results of single and three pulse photoselection studies of the dynamics of a fluorescent probe (Oxazine 4) in the nematic phase of the liquid crystal 4-n-pentyl-4'-cyanobiphenyl (5CB). The combination of these techniques with time resolved fluorescence anisotropy measurements allows the unambiguous determination of the full angular motion of the probe together with the ground and excited state degrees of equilibrium (steady state) alignment. The restricted geometry imposed by the nematic host is seen to impart a marked anisotropy in theta and phi diffusion (gamma(phi)/gamma(theta)similar to4)