Multiphoton imaging time

P.E. Morton, T.C. Ng, S.A. Roberts, B. Vojnovic, S.M. Ameer-Beg, Time resolved multiphoton imaging of the interaction between the PKC and NFkB signalling pathways, Proc. SPIE 5139, 216-222 (2003)... 

Fig. 4.1. Multiphoton fluorescence intensity (A-C) and TCSPC fluorescence lifetime images (D-F) of fresh unstained sections of human cervical biopsy excited at 740 nm and imaged between 385 and 600 nm. The individual acquisition times were 600 s. Adapted from Fig. 22.11 of Ref. [8].

The introduction and diversification of genetically encoded fluorescent proteins (FPs) [1] and the expansion of available biological fluorophores have propelled biomedical fluorescent imaging forward into new era of development [2], Particular excitement surrounds the advances in microscopy, for example, inexpensive time-correlated single photon counting (TCSPC) cards for desktop computers that do away with the need for expensive and complex racks of equipment and compact infrared femtosecond pulse length semiconductor lasers, like the Mai Tai, mode locked titanium sapphire laser from Spectra physics, or the similar Chameleon manufactured by Coherent, Inc., that enable multiphoton excitation. 

Greenhalgh, C., Cisek, R., Prent, N., Major, A., Aus der Au, J., Squier, J., and Barzda, V. 2005. Time and structural image analysis of microscopic volumes, simultaneously recorded with second harmonic generation, third harmonic generation, and multiphoton excitation fluorescence microscopy. Proc. SPIE 5969 59692F1-F8. 

In addition to using imaging as a technique to obtain spatially and temporally patterned chemical data from a sample, one can also pattern chemical reactions in space and time using similar methods. Figure 2.18 demonstrates an example in which multiphoton scanning with ultrafast laser pulses was used to polymerize a photoresist resin with about 120 nm spatial resolution in three dimensions. 

Figure 24. Coincidence-imaging spectroscopy of dissociative multiphoton ionization processes in NO2 with 100-fs laser pulses at 375.3 nm, using angle-angle correlations. The polar plots show, at time delays of Ofs, 350 fs, 500 fs, 1 ps, and 10 ps, the angular correlation between the ejected electron and NO photofragment when the latter is ejected parallel to the laser field polarization vector. The intensity distributions change from a forward-backward asymmetric distribution at early times to a symmetric angular distribution at later times, yielding detailed information about the molecule as it dissociates. Taken with permission from Ref. [137]...

Thus, multiphoton excitation eliminates unwanted out-of-focus excitation, unnecessary phototoxity and bleaching. However, efficient power sources are required and, since the efficiency of multiphoton excitation is usually low, the times needed to generate images are increased. 

Another important aspect about the optical properties of QDs is the multiphoton process which has been widely applied in recent years in biological and medical imaging after the pioneer work of Goeppert-Mayer (1931), Lami et al. (1996), Helmchen et al. (1996), Yokoyama et al. (2006). The multiphoton process has largely been treated theoretically by steady-state perturbation approaches, for example, the scaling rules of multiphoton absorption by Wherrett (1984) and the analysis of two-photon excitation spectroscopy of CdSe QDs by Schmidt et al. (1996). Non-perturbation time-dependent Schrodinger equation was solved to analyze the ultrafast (fs) and ultra-intense (in many experiments the optical power of laser pulse peak can reach... 

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