Two photon fluorescence

Figure Bl.1.4. Two-photon fluorescence excitation spectrum of naphthalene. Reprinted from [35], Courtesy, Tata McGraw-Hill Publishing Company Ltd, 7 West Patel Nagar, New Dehli, 110008, India.

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... 

Kim Ki H, So P T C, Kochevar I E, Masters B R and Gratton E 1998 Two-photon fluorescence and confocal reflected light imaging of thick tissue structures Proc. SPIE 3260 46-57... 

The example we consider is the two-photon fluorescence excitation specfrum of 1,4-difluorobenzene, shown in Figure 9.29 and belonging to the >2 point group. The transition between the ground and first singlet excited state is Table A. 3 2 in Appendix A shows that 82 = r(T ) and, therefore, according to Equation (7.122), the electronic transition is allowed as a one-photon process polarized along the y axis which is in-plane and... 

Figure 9.29 Two-photon fluorescence excitation spectrum of 1,4-difluorobenzene. The upper and lower traces are obtained with plane and circularly polarized radiation, respectively, but the differences are not considered here. (Reproduced, with permission, Ifom Robey, M. J. and Schlag, E. W., Chem. Phys., 30, 9, 1978)...

Nevertheless, 1,4-difluorobenzene has a rich two-photon fluorescence excitation spectrum, shown in Figure 9.29. The position of the forbidden Og (labelled 0-0) band is shown. All the vibronic transitions observed in the band system are induced by non-totally symmetric vibrations, rather like the one-photon case of benzene discussed in Section 7.3.4.2(b). The two-photon transition moment may become non-zero when certain vibrations are excited. 

A nano-light-source generated on the metallic nano-tip induces a variety of optical phenomena in a nano-volume. Hence, nano-analysis, nano-identification and nanoimaging are achieved by combining the near-field technique with many kinds of spectroscopy. The use of a metallic nano-tip applied to nanoscale spectroscopy, for example, Raman spectroscopy [9], two-photon fluorescence spectroscopy [13] and infrared absorption spectroscopy [14], was reported in 1999. We have incorporated Raman spectroscopy with tip-enhanced near-field microscopy for the direct observation of molecules. In this section, we will give a brief introduction to Raman spectroscopy and demonstrate our experimental nano-Raman spectroscopy and imaging results. Furthermore, we will describe the improvement of spatial resolution... 

Figure 8.5 Transmitting (a) and two-photon fluorescence (b) images of a part of Zygnema in water. Scale bar is 5 tm in length.

Agate, B., Brown, C., Sibbett, W. and Dholakia, K. (2004) Femtosecond optical tweezers for in-situ control of two-photon fluorescence. Opt. Express, 12, 3011-3017. 

Fig. 6 Normalized linear absorption (solid black line), anisotropy (blue circles), and corresponding 2PA spectrum measured by two-photon fluorescence (red squares) and Z-scan (green circles) for SD 2405. Molecular structure is shown to the left...

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. 

At a fundamental wavelength of 1064 nm, large and anisotropic optical nonlinearity was also observed. The values of X (3) and are 4.5 x 1010 esu and 1.0 x 10 10 esu, respectively. The large values are due to the two photon resonance, because the harmonic wavelength of 355 nm is near off-resonance region. From the two-photon fluorescence measurement, we confirmed that a two-photon absorption band, which is origin of the enhancement effect, exist around 532 nm, half of the fundamental wavelength. 

Chen Y., Muller J. D., Eid J. S. and Gratton E. (2001) Two-Photon Fluorescence Fluctuation Spectroscopy, in Valeur B. and Brochon J. C. (Eds) New Trends in Fluorescence Spectroscopy. Applications to Chemical and Life Sciences, Springer-Verlag, Berlin, pp. 276-96. 

So P. T. C., French T., Yu W. M., Berland K. M., Dong C. Y. and Gratton E. (1996) Two-Photon Fluorescence Microscopy Time-Resolved and Intensity Imaging, in Wang X. F. and Herman B. (Eds), Fluorescence Imaging Spectroscopy and Microscopy, Chemical Analysis Series, Vol. 137, John Wiley Sons, New York, pp. 351-74. 

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. 

Hanson KM, Behne MJ, Barry NP, Mauro TM, Gratton E, Clegg RM (2002) Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient. BiophysJ 83 1682-1690. 

Guild, J. B., and Webb, W. W. 1995. Line scanning microscopy with two-photon fluorescence excitation. Biophys. J. 68 290a. 

Hanson, K. M., and Clegg, R. M. 2005. Two-photon fluorescence imaging and reactive oxygen species detection within the epidermis. Methods Mol. Biol. 289 413-22. 

Hopt, A., and Neher, E. 2001. Highly nonhnear photodamage in two-photon fluorescence microscopy. Biophys. J. 80 2029-36. 

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