Fluorescence depletion technique

Watanabe, T., Iketaki, Y., Omatsu, T., Yamamoto, K., Sakai, M. and Fujii, M. (2003) Two-point-separation in superresolution fluorescence microscope based on up-conversion fluorescence depletion technique. Opt. Express, 11, 3271-3276. 

Figfure 6. Sequence of measurements required for the fluorescence depletion technique (FRA). Shown is the complete data set for erythrosin-5 -isothiocyanate in a solid matrix of polymethyl- methacrylate. In traces (A) -(C) the parallel (to the actinic laser) [channels 1-512] and perpendicular [channels 513-1024] emission components are shown for (A) depletion signals (both lasers active) (B) excimer (actinic) laser only (C) steady-state fluorescence (i.e. with only the continuous wave argon laser) (D) polarized emission components after subtraction... 

As indicated earlier, only probes with relatively high values of isc suitable for phosphorescence emission measurements. The triplet state of fluorescein (see Table II) is not detectable by phosphorescence emission techniques but should provide a suitable probe for the fluorescence depletion technique (11,15,16). Figure 8 shows FRA data for the rotational diffusion at 6 C and in 98%... 

We wish to reveal the mechanism for the optimal dynamic discrimination between the very similar biochromophores riboflavin (RBF) and flavin mononucleotide (FMN) using optimally shaped laser fields. Our FISH simulations utilize experimentally optimized laser fields and show that the fluorescence depletion ratio between two molecules can be manipulated with such fields, eventually achieving discrimination between them. Moreover, these results validate for the first time the experimental optimal control technique applied on complex systems [63]. 

Because they lend themselves to studies using both photochemical and chemical activation, bimolecular reactions of vibrationally excited hydrogen halides have been more throughly investigated than any other family of reactions. The rate constants in Table 1.3 have been obtained by the laser-induced vibrational fluorescence technique and correspond to the sum of rate constants for reactive and inelastic processes. The main problem is to establish the atomic concentrations accurately. This is usually accomplished by gas-phase titration in a discharge-flow system, although photolysis methods have also been employed. To find the ratio of reaction to non-reactlve relaxation, product concentrations have to be observed. This has been done in relatively few cases. Some systems have also been studied using the infrared chemiluminescence depletion technique (see Section 1.5.1). These experiments supply relative rate data for removal from several vibrational levels, and, in favorable cases, also provide some information about the rotational-state dependence of these rates. 

Chemical analysis of the metal can serve various purposes. For the determination of the metal-alloy composition, a variety of techniques has been used. In the past, wet-chemical analysis was often employed, but the significant size of the sample needed was a primary drawback. Nondestmctive, energy-dispersive x-ray fluorescence spectrometry is often used when no high precision is needed. However, this technique only allows a surface analysis, and significant surface phenomena such as preferential enrichments and depletions, which often occur in objects having a burial history, can cause serious errors. For more precise quantitative analyses samples have to be removed from below the surface to be analyzed by means of atomic absorption (82), spectrographic techniques (78,83), etc. 

Fourier transform infrared (FTIR) spectroscopy, 13C nuclear magnetic resonance (NMR) spectroscopy, ultraviolet-visible (UV-VIS) and fluorescence spectroscopy can be integrated with chromatographic techniques especially in the study of ageing and degradation of terpenic materials. They can be used to study the transformation, depletion or formation of specific functional groups in the course of ageing. 

Much larger RISC values were found for the cyanine dyes 56-58 [38]. A similar two-color technique was used to measure these yields with the exception that meyo-tetraphenylporphyrin was used in place of Aberchrome 540 as the two-laser actinometer. As Table 4 shows, there is an inverse relationship between the triplet depletion (bleaching) quantum yield, 4>B1, and the 5 —> T ISC yield, 1SC. Thus a large value for 4>B1 was accompanied by a small 4>ISC. This reflects the cyclic flow of energy following excitation of Tx to Tn. Once RISC has occurred, decay of the S, state partitions between ISC and fluorescence. If 4>ISC is small, relatively few Sj states will be cycled back to T and 4>B1 will be large. This effect masks the actual efficiency of RISC. Thus the ratio < B1/(1 — isc) s giyen as 311 indicator of RISC efficiency. 

Much of the early studies of surfactant adsorption at the solid-solution interface were based on classical experimental techniques, such as solution depletion [1, 32], fluorescence spectroscopy [2], and measurements of the differential enthalpy of adsorption [2], Such methods have provided much of the basic initial understanding. However, they provide no direct structural information and are difficult to apply to mixtures [23, 34], However, when combined with other techniques, such as NMR and flow microcalorimetry, they provide some insight into the behaviour of mixtures. This was demonstrated by Thibaut et al. [33] on SDS/C10E5 mixtures adsorbed onto silica and by Colombie et al. [34] on the adsorption of SLS/Triton X-405 mixtures onto polystyrene particles. 

Figure 12-1. Schematic diagram to illustrate double resonance techniques, (a) REMPI 2 photon ionization. The REMPI wavelength is scanned, while a specific ion mass is monitored to obtain a mass dependent SI <- SO excitation spectrum, (b) UV-UV double resonance. One UV laser is scanned and serves as a burn laser, while a second REMPI pulse is fired with a delay of about 100 ns and serves as a probe . The probe wavelength is fixed at the resonance of specific isomer. When the burn laser is tuned to a resonance of the same isomer it depletes the ground state which is recorded as a decrease (or ion dip) in the ion signal from the probe laser, (c) IR-UV double resonance spectroscopy, in which the burn laser is an IR laser. The ion-dip spectrum reflects the ground state IR transitions of the specific isomer that is probed by the REMPI laser, (d) Double resonance spectroscopy can also use laser induced fluorescence as the probe, however that arrangement lacks the mass selection afforded by the REMPI probe...

Negative adsorption is in most cases very small compared to positive adsorption and therefore not easily detected directly. A sophisticated optical technique (evanescent-wave-induced fluorescence, EWIF) has been used to prove the reduction in segment concentration close to a non-adsorbing surface Depletion also has an effect on the flux of polymer solutions through pores since the viscosity of the liquid near the non-adsorbing surface is lower than that of the polymer solution, the flux is then higher than would be expected on the basis of the bulk viscosity. Negative adsorption at a liquid-air interface leads to a measurable increase In surface tenslon ... 

Several far-field light microscopy methods have recently been developed to break the diffraction limit. These methods can be largely divided into two categories (1) techniques that employ spatially patterned illumination to sharpen the point-spread function of the microscope, such as stimulated emission depletion (STED) microscopy and related methods using other reversibly saturable optically linear fluorescent transitions (RESOLFT) [1,2], and saturated structured-illumination microscopy (SSIM) [3], and (2) a technique that is based on the localization of individual fluorescent molecules, termed Stochastic Optical Reconstruction Microscopy (STORM [4], Photo-Activated Localization Microscopy (PALM) [5], or Fluorescence Photo-Activation Localization Microscopy (FPALM) [6]. In this paper, we describe the concept of STORM microscopy and recent advances in the imaging capabilities of STORM. 

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