Two-photon confocal microscopy

Brakenhoff G J, Squier J, Norris T, Bliton A C, Wade M FI and Athey B 1996 Real-time two-photon confocal microscopy using a femtosecond, amplified Ti sapphire system J. Microscopy 181 253-9... 

Multifunctional materials will play an important role in the development of Photonics Technology. This paper describes novel multifunctional polymeric composites for applications in both active and passive photonic components. On the molecular level, we have introduced multifunctionality by design and synthesis of chromophores which by themselves exhibit more than one functionality. At the bulk level, we have introduced the concept of a multiphasic nanostructured composites where phase separation is controlled in the nanometer range to produce optically transparent bulk in which each domain produces a specific photonic function. Results are presented from the studies of up-converted two-photon lasing, two-photon confocal microscopy, optical power limiting, photorefractivity and optical channel waveguides to illustrate the application of the multifunctional optical composites. 

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 or Two-Photon Confocal Microscopy... 

FCS is a type of spectroscopy that is based on the measurement of fluorescence intensity and the analysis of its fluctuations, which result from diffusion of the observed fluorophore in the excitation volume or changes in the fluorescence quantum yield arising from chemical reactions. Measurements are usually made on only a few molecules at a time, of the order of 10 milliseconds. This is achieved by illuminating tiny sample volumes ( 1 femtoliter) by employing two-photon excitation microscopy (see section Two-Photon Confocal Microscopy above). FCS is used to measure translational diffusion coefficients of macromolecules, the number of fluorescent molecules under observation and the... 

Bowman R.D., Kneas K.A., Demas J.N., Periasamy A., Conventional, Confocal And Two-Photon Fluorescence Microscopy Investigations of Polymer-Supported Oxygen Sensors, J. Microscopy 2003 211 112-120. 

For samples thicker than the depth of field, the images are blurred by out-of-focus fluorescence. Corrections using a computer are possible, but other techniques are generally preferred such as confocal microscopy and two-photon excitation microscopy. It is possible to overcome the optical diffraction limit in near-field scanning optical microscopy (NSOM). 

Two-photon fluorescence microscopy has also been used with good effect in the near-IR. For example, Ferguson et al.r24> at the University of Strathclyde have used 270 fsec pulses from a titanium sapphire (Ti sapphire) laser at 790 nm to observe visible fluorescence from dyes in zebra fish larvae and erythrocytes. The high depth and lateral definition afforded by the two-photon process and confocal microscopy are useful here. Also, the use of near-IR excitation minimizes photobleaching. 

Figure 11.12 A comparison between confocal and two-photon fluorescence microscopy in confocal microscopy fluorescence is excited throughout the sample while in two-photon excitation it is restricted to the confocal region around the focal point of the laser...

Lindek St, Cremer Chr and Stelzer E H K 1996 Confocal theta fluorescence microscopy using two-photon absorption and annular apertures Optik 02 131-4... 

Bhawalkar J D, Swiatkiewicz J, Pan S J, Samarabandu J K, Liou W S, He G S, Berezney R, Cheng P C and Prasad P N 1996 Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore Scanning 18 562-6... 

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],... 

Diaspro A, Chirico G, Federici F, Cannone F, Beretta S, Robello M (2001) Two-photon microscopy and spectroscopy based on a compact confocal scanning head. J Biomed Opt 6 300-310... 

Lifetime imaging can be implemented both in wide field and in scanning microscopes such as confocal microscopes and two-photon excitation microscopes. The most common implementations in time-domain fluorescence lifetime imaging microscopy (FLIM) are based on TCSPC [8, 9] and time-gating (TG) [2, 10],... 

As mentioned above, spectral imaging microscopy is a form of multidimensional fluorescent microscopy where a fluorescent emission spectrum is acquired at each coordinate location in the sample. This mode of imaging has been implemented for wide field, confocal, and two-photon laser scanning microscopy, and several excellent... 

Two-photon excitation provides intrinsic 3-D resolution in laser scanning fluorescence microscopy. The 3-D sectioning effect is comparable to that of confocal microscopy, but it offers two advantages with respect to the latter because the illumination is concentrated in both time and space, there is no out-of-focus photo-bleaching, and the excitation beam is not attenuated by out-of-focus absorption, which results in increased penetration depth of the excitation light. 

Finally, in Chapter 11 some advanced techniques are briefly described fluorescence up-conversion, fluorescence microscopy (confocal excitation, two-photon excitation, near-field optics, fluorescence lifetime imaging), fluorescence correlation spectroscopy, and single-molecule fluorescence spectroscopy. 

The two cationic dyes, Py+ as a donor and Ox" as an acceptor, were found to be very versatile for demonstrating photonic antenna functionalities for light harvesting, transport, and capturing, as illustrated in Fig. 7. They can be incorporated into zeolite L by means of ion exchange, where they are present as monomers because of the restricted space. In this form they have a high fluorescence quantum yield and favourable spectral properties. The insertion of the dyes can be visualised by means of fluorescence microscopy. The fluorescence anisotropy of Ox -loaded zeolite L has recently been investigated in detail by conventional and by confocal microscopy techniques [15],... 

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