Single photon confocal microscopy

Fig. 15 Zinc salen complexes and colocalisation in endoplasmic reticulum via single-photon confocal microscopy before and after irradiation for 315 s. ...

For example, the Rayleigh scattering of UV wavelength photons is a particular problem in single-photon confocal microscopy [17]. Furthermore, another point to note is that the emission wavelength for MPM is substantially shorter than the excitation wavelength, which makes for easier differentiation and hence detection. 

Two-Photon Confocal Microscopy. Confocal microscopy provides the possibilities of optical sectioning of a sample by the use of a spatial filter to improve axial resolution of the optical microscope (20). These optical sections can be used to reconstruct the 3D structure of a polymeric specimen. Confocal microscopy using fluorescence is usually performed by a single-photon excitation (linear absorption) of a fluorophore which may be chemically an integral part of the system (polymer) being investigated or just... 

Two-photon excited fluorescence detection at the single-molecule level has been demonstrated for cliromophores in cryogenic solids [60], room-temperature surfaces [61], membranes [62] and liquids [63, 64 and 65]. Altliough multiphoton excited fluorescence has been embraced witli great entluisiasm as a teclmique for botli ordinary confocal microscopy and single-molecule detection, it is not a panacea in particular, photochemical degradation in multiphoton excitation may be more severe tlian witli ordinary linear excitation, probably due to absorjDtion of more tlian tire desired number of photons from tire intense laser pulse (e.g. triplet excited state absorjDtion) [61],... 

Perhaps the most significant contrihution that two-photon excited fluorescence has made in biological research to date is in the area of fluorescent imaging. Prior to the invention of two-photon microscopy in 1990, depth resolution in fluorescence microscopy was achieved using the confocal microscope, the principles of which are illustrated in Figure 11.9. Fluorescence is excited via single-photon excitation... 

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...

The combination of illuminating the sample with a strongly focused laser beam and observing the laser-induced fluorescence or the non-resonant scattered laser light with confocal microscopy allows the detection of single molecules and their diffusion through a liquid medium. Each molecule can emit N = T/2r photons (see Fig. 10.2) where T is the diffusion time through the laser beam and r the upper state lifetime. 

The main luminescence parameters traditionally measured are the frequency of maximal intensity Vmax, intensity I, the quantum yield < >, the hfetime of the exited state T, polarization, parameters of Raman spectroscopy, and excited-state energy migration. The usefulness of the fluorescence methods has been greatly enhanced with the development of new experimental techniques such as nano-, pico-, and femtosecond time-resolved spectroscopy, single-molecule detection, confocal microscopy, and two-photon correlation spectroscopy. 

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