Ground state depletion

Hell S W and Kroug M 1995 Ground-state-depletion fluorescence microscopy a concept for breaking the diffraction resolution limit Appl. Phys. B 60 495-7... 

As in the case of most FRET imaging formalisms, Eq. (12.4) applies to the donor at a particular x, y, x coordinate j as a virtual species with an apparent kt(j) it applies for arbitrary absolute and relative local concentrations of donor and acceptor but only if population distributions are properly considered (see other chapters). For example, assuming a population of donors, a fraction a of which are in a unique DA environment (e.g., as a bound complex) and the rest free, one can generate an expression for a in terms of known parameters and experimental signals (varying from point to point), kt/kf, Qd, and Qa- Related expressions can be formulated in terms of donor and acceptor emission anisotropies and lifetimes, and for conditions of nonlinearity such as ground state depletion of the donor and/or acceptor [1] (see Section 12.3). 

The direct detection of the S <- Sj absorption in organic compounds has so far been achieved by a nanosecond or picosecond laser flash photolysis method. The general features of transient absorption spectra of metalloporphyrins actually suggest the presence of strong absorption bands in visible or ultraviolet region (38-40). However, as the transient absorption of the state often overlaps with that of ground state depletion, it is usually difficult to evaluate the absolute absorption cross sections for the transition by... 

A more complicated solution (Equation 6.17) results when Equation 6.15 is integrated after the incorporation of the ground-state depletion described by Equation 6.16. 

The ground-state depletion is more commonly observed in photochemical experiments such as flash photolysis than the simultaneous biphotonic absorption. Another common deviation from the Beer s law due to the sequential absorption of two photons will be discussed later in this section. 

J. Foiling et ah. Fluorescence nanoscopy by ground-state depletion and singlemolecule return. Nat. Meth. 5, 943-945 (2008)... 

All these studies with femtosecond pulses on the primary photochemical processes of rhodopsin were done by means of transient absorption (pump probe) spectroscopy [10]. However, absorption spectroscopy may not be the best way to probe the excited-state dynamics of rhodopsin, because other spectral features, such as ground-state depletion and product absorption, are possibly superimposed on the excited-state spectral features (absorption and stimulated emission) in the obtained data. Each spectral feature may even vary in the femtosecond time domain, which provides further difficulty in analyzing the data. In contrast, fluorescence spectroscopy focuses only on the excited-state processes, so that the excited-state dynamics can be observed more directly. 

The nonlinear behavior of the light output was originally ascribed to ground state depletion of the activator. In sulfides, where the activator concentration is low ( 0.01%), this is certainly important. Therefore, attention shifted to the oxidic phosphors where the activator concentrations are much higher ( 1 %). However, excited state absorption and Auger processes (Sect. 4.6) will also result in saturation effects. Detailed analysis of interactions between excited activator ions are available [9,10]. 

Absorbance difference spectra of H. chlorum membrane fragments upon 532 nm excitation at 15 K are shown in Fig.l. The difference spectmm measured at 40 ps after the excitation flash shows negative bands near 665, 793 and 812 nm. At 350 ps sifter the flash the amplitude of the 812 nm band is strongly reduced. We ascribe this band to ground-state depletion of Bchl g 808 by the formation of singlet excited states. The band near 793 nm is attributed to photooxidation of P758, the primary donor. 

In hole-burning spectroscopy, on the other hand, the bum laser frequency is fixed while the probe laser frequency is tuned. The pump laser depletes the ground state of a single conformation, so that the probe laser records an excitation spectmm of all conformers except the one selected by the bum laser. It should be noted that in both cases the hot bands are not affected, because only the ground state is depopulated. In addition, for both methods to be effective, the bum laser pulse energy should be sufficient to induce a substantial ground state depletion. 

Identical formulae apply in both cases. When excited molecules do not rotate or transfer excitation energy to differently oriented molecules between the initial absorption event and the later absorption (photoinduced dichroism) or emission (photoluminescence) event, their orientation factors are time independent. Negligible ground-state depletion is assumed in the first step. 

Stochastic optical reconstruction microscopy (STORM) and direct STORM (dSTORM), also referred to as ground-state depletion and single-molecule return (GSDIM), use fluorochromes that reversibly cycle between fluorescent (ON) and dark (OFF) states upon exposure to light of specific wavelengths [158, 162-164]. STORM-based ICC (Fig. 3D) relies on the proximity of two fluorochromes attached to an antibody in a specific ratio and at a specific distance (Fig. 3E), whereas dSTORM (Fig. 3E) employs a... 

FoUing J, Bossi M, Bock H et al (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule retmn. Nat Methods 5 943-945... 

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