Fluorescence stimulated emission depletion

One way to deplete the fluorescence, stimulated emission depletion (STED) microscopy, is to return the excited fluorophores to the ground state by stimulated emission as we discussed above in connection with temporal resolution of Stokes shifts (Sect. 5.2). Another approach is to use a variant of GFP that can be switched between fluorescent and non-flourescent states by light of different wavelengths (Sect. 5.7). This switching can be achieved with lower light intensities than are needed to induce stimulated emission [149, 229]. The essential requirements in either case are for the depletion pulse intensity to be zero at or near the focus of the excitation, and for the depletion process to approach saturation rapidly as the pulse intensity increases. 

Hell, S. W. and Wichmann, J. (1994). Breaking the Diffraction Resolution Limit by Stimulated-Emission—Stimulated-Emission-Depletion Fluorescence Microscopy. Opt. Lett. 19, 780-2. 

Fig. 19.4. Stimulated emission depletion (STED) microscopy reveals densely packed charged nitrogen vacancy (NV) color centers in a diamond crystal, (a) State diagram of NV centers in diamond (see inserted sketch) showing the triplet ground ( A) and fluorescent state ( E) along with a dark singlet state ( E) and the transitions of excitation (Exc), emission (Em), and stimulated emission (STED). (b) The steep decline in fluorescence with increasing intensity /sted shows that the STED-beam is able to switch off the centers almost in a digital-like fashion. This nearly rectangular ...

S.W. HeU, Improvement of lateral resolution in far-Held light microscopy using two-photon excitation with offset beams. Opt. Commun. 106, 19-24 (1994) S.W. HeU, J. Wichmann, Breaking the diffraction resolution limit by stimulated emission stimulated emission depletion fluorescence microscopy. Opt. Lett. 19(11), 780-782 (1994)... 

T.A. Klar et al., Fluorescence microscopy with diffraction resolution limit broken by stimulated emission. Proc. Natl. Acad. Sci. U.S.A 97, 8206-8210 (2000) T.A. Klar, E. Engel, S.W. Hell, Breaking Abbe s diffraction resolution limit in huorescence microscopy with stimulated emission depletion beams of various shapes. Phys. Rev. E 64, 066613, 1-9 (2001)... 

B. Hein, K. WiUig, S.W. HeU, Stimulated emission depletion (STED) nanoscopy of a fluorescent protein - labeled organelle inside a living cell. Proc. Natl. Acad. Sci. USA 105(38), 14271-14276 (2008)... 

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. 

S.W. Hell, J. Wichmami, Breaking the diffraction resolution limit by stimulated emission stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780-782 (1994)... 

Figure 11.21 StimuLated emission depletion (STED) microscopy. The sample is excited using single-photon excitation (PUMP pulse) in a confocal microscope arrangement. A time-delayed DUMP pulse selectively depletes close to 100% of the exdted state population in a region around the focus of the PUMP pulse. Using this approach. Hell and co-workers were able to obtain a 5-fold reduction in the fluorescent spot size in the vertical (Z-direction) and a greater than a 2-fold reduction in the horizontal Y/X) direction, leading to a final image size of 97 by 104 nm...

Stimulated emission depletion (STED) microscopy is another microscopy approach to overcome the diffraction limitation of light microscopy by inhibiting fluorescence outside of the focal spot [9]. In this context, fluorescence is viewed as spontaneous light emission while stimulating emission via synchronized laser... 

Ear-field nanoscopic measuring technique Ear-field optical nanoscopic measuring technique Laser-induced fluorescence photobleaching anemometer Nanoscopy Stimulated emission depletion... 

To realize the observation/detection on nanofluidics, the LIF with high resolution is a vital factor. With the release of stimulated emission depletion (STED) fluorescence microscope, the super-resolution LIF microscope on confocal manner, the high resolution at the order of nanometer was obtained [7, 8]. It was utilized to measure the velocity of fluorescent probes in nanofluidics and to observe the ion... 

Stimulated emission depletion microscopy (STED—Fig. 3F) is based on a process called stimulated emission whereby fluorescent molecules are effectively switched OFF at the edge of a laser spot, thereby allowing only the fluorochromes at the very center to fluoresce [172]. STED permits achieving fluorescence nanos-copy that revealed neurons in the mouse cerebral cortex at resolution below 70 nm [173]. Other examples of STED applications can be found in [174-178]. 

For many fluorescence-based imaging techniques, such as confocal laser scanning microscopy, spinning disk confocal microscopy, 4pi microscopy, stimulated emission depletion (STED) microscopy, and other superresolution microscopy techniques, we refer to the indicated literature. 

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. 

Finally in figure 13c we depict the scheme for stimulated emission pumping, which is the stimulated equivalent of dispersed LIF. I Jhen the probe is on resonance this stimulates emission in the direction of the probe laser beam, thereby depleting spontaneous fluorescence. The resonance condition can therefore be sought through detection of fluorescence side-light without the losses inherent in the use of a monochromator. 

A method, femtosecond time-resolved stimulated emission pumping (SEP) fluorescence depletion (FS TR SEP ED), has been developed to study the vibrational relaxation of electronic excited states of molecules (Figures 11.9 and 11.10) [31]. 

Fig. 9 Schematic illustration of STED. (a) Jablonski diagram indicating the So and Si state of a molecule (left). A femtosecond pulse excites the fluorophore from So to a high vibrational level in Si. The following slow STED pulse induces the stimulated emission from the lowest Si level to a high vibrational level in So. The STED pulse depletes the Si state and quenches the fluorescence. The corresponding wavelength diagram is shown on the right-hand side the dotted and solid lines are the absorption and emission spectra, respectively. The spontaneous emission at A-oet is detected as the signal in the microscopy measurement, (b) The combination of the diffraction-limited excitation spot and the engineered STED pulse yields a narrowed point spread function...

Zhong, Q.H., Wang, Z.H., Sun, Y., Zhu, Q.H., Kong, F.N. Vibrational relaxation of dye molecules in solution studied by femtosecond time-resolved stimulated emission pumping fluorescence depletion. Chem. Phys. Lett. 248, 277-282 (1996)... 

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