Two-photon fluorescence technique

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... 

Due to the need to use expensive and complicated laser pump systems, early development occurred primarily by physicists. Two-photon absorption properties of existing materials were extensively studied in this stage. In early 1990s, some potential apphcations with this technique were demonstrated, such as two-photon fluorescence (2PF) 3D data storage [2] and 2PF microscopy [3]. However, the 2PA efficiency of the materials is the bottleneck for these applications, and this has triggered the effort to search for new materials with higher... 

Whether the components of the gene carriers actually remain associated during import into the nucleus or enter individually cannot be answered by optical methods as their resolution is limited. A possible technique to study the complexation of DNA within cells is fluorescence correlation spectroscopy (FCS). Clamme et al. studied the intracellular fate of PEI after transfection with polyplexes by two-photon fluorescence FCS [54]. They showed that PEI binds to the inner membrane of endosomes and lysosomes and shows free diffusion in the cytosol as well as the nucleus. However, they did not detect any PEI/DNA complexes inside the nucleus. 

If intrinsic two-photon absorption cross-sections are the subject of investigation short pulses (ps or fs) should be used and two-photon fluorescence (see below) should be measured to verify the Independence of the TPA process. Most often, two-photon absorption is measured either with the Z-scan technique (see above) or with the nonlinear transmission method [38]. 

In TPA induced fluorescence spectroscopy the excited state population for fluorescence is created by the simultaneous absorption of two photons instead of the one-photon absorption in standard fluorescence experiments. The principles of this technique are described in Ref. [23]. It allows an experimental determination of two-photon absorption cross-sections provided the material is fluorescent and that its two-photon fluorescence quantum efficiency is known (which is usually assumed to be equal to the one-photon fluorescence quantum efficiency if the same excited state is reached). In this case the method generally provides high sensitivity. 

However, as we noted earlier (in Section II), it is most important to have a full temporal and spatial profile of the picosecond laser pulses in order to correctly interpret the one or two photon excited molecular response functions (absorption, emission, Raman scattering) of the system under study. While autocorrelation techniques such as second harmonic generation (SHG) or two photon fluorescence have always been the route to such short pulse measurements, a significant advance has recently been made in bringing these conventional autocorrelation measurements into the real-time domain. ... 

TPA can be measured by the transmission method or by the Z-scan technique. Moreover, two-photon fluorescence can serve to measure TPA absorption cross-sections, provided that a fluorescent excited state is reached by TPA. In nonlinear... 

A series of new ( )-4,4 -bis(diphenylammo)stilbene derivatives have been synthesized to investigate nonlinear absorptivities with attention paid to the peripheral substituent effect and multibranched modification effect by the open aperture femtosecond Z-scan technique and the nanosecond nonlinear optical transmission (NET), respectively [36]. Two-photon fluorescence for ( )-4,4 -bis(di-phenylamino)stilbene in THE was detected. It was found that substituent group attached to the periphery of BDPAS has no obvious contribution to TPA enhancement and that the dramatic increase in effective TPA cross sections of multibranched samples in nanosecond regime strongly suggested their larger excited-state absorption. 

It has been suggested that this increase in nonlinearity may be an artifact (two-photon fluorescence) of the hyperpolarizability measurement technique (hyper-Rayleigh scattering). Studies are currently under way at the University of Southern California (USC) to resolve this question, by using both electric field-induced... 

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