Multi-photon microscopy

Deguil, N., Mottay, E., Salin, F., Legros, P. and Choquet, D. (2004). Novel diode-pumped infrared tunable laser system for multi-photon microscopy. Microsc. Res. Tech. 63, 23-6. 

In contrast to the other listed single molecule techniques, measurements based on fluorescence correlation spectroscopy (FCS) can already be performed both routinely and rapidly. Moreover, FCS is applied in many scientific disciplines and the number of applications of this technique is growing very rapidly. Thus, its principles will be briefly outlined Usually, a sharply focused laser beam illuminates a volume element of about 10 1 by using confocal or multi-photon microscopy. 

Multi-photon microscopy for in situ characterization is a fluorescence laser scanning technique (see Chapter 8). This technique can produce 3D images, which can be stacked to get time series for different interesting phenomena in UF and MF. Fouling inset can be characterized and thus also the critical flux can be measured. Fouling of membranes, especially by proteins, has been studied with this technique. Because the target substance needs to be fluorescent, not all kinds of filtration processes can be monitored in this way. 

Optical clearing seems to be a promising technique for improvement of deteeted signals in multi-photon microscopy and nonlinear spectroscopy and imaging of tissues [6, 61, 100]. From the other hand these techniques might be useful in understanding of molecular mechanisms of tissue optical clearing at immersion and dehydration [61, 100]. 

Following this initial report, several groups have used already existing complexes for multi-photon microscopy [62,88,103,104]. All these complexes, described in Fig. 5.15, are stable and soluble in aqueous media. Furthermore, they are able to stain cells and do not present significant cell toxicity. But, since they were not especially designed for two-photon application (no CT excited state is present in the sensitising chromophore), their unoptimised two-photon cross-sections are low (<10GM). 

Barber, P. R., Ameer-Beg, S. M., Gilbey, J. D., Edens, R. J., Ezike, I. and Vojnovic, B. (2005). Global and pixel kinetic data analysis for FRET detection by multi-photon time-domain FLIM. In Multiphoton Microscopy in the Biomedical Sciences V.Vol. 5700. SPIE, San Jose, CA, USA, pp. 171-81. 

Kawata, Y, Xu, C., and Denk, W. 1999. Feasibility of molecular-resolution fluorescence nearfield microscopy using multi-photon absorption and field enhancement near a sharp tip. J. Appl. Phys. 85 1294-1301. 

Masters BR, So PTC. Multi-photon excitation microscopy and confocal microscopy imaging of in vivo human skin a comparison. Microscopy and Microanalysis 1999, 5, 282-289. 

Fluorescence microscopy, which has been applied by Jain and co-workers in their studies of interstitial diffusion [20, 21] and lymphatic flow, can be used for measurements within the tissue of a living animal, provided that the tissue can be accessed by light. This access can sometimes be obtained by installing window chambers in the tissue [22]. Multi-photon fluorescence imaging, an important new technique introduced by Webb and colleagues [23-25], promises to broaden the applications of this technique, since quantitative fluorescence imaging can be performed in three-dimensional specimens, even specimens that scatter light. 

Park, Y.I., et al. Upconverting nanoparticles a versatile platform for wide-field two-photon microscopy and multi-modal in vivo imaging. Chemical Society Reviews 44, 1302-1317 (2015)... 

Consequently, the main supphers of commercial fluorescence microscopes never seriously considered producing dedicated instrumentation for TRFM. In the past two decades, however, confocal microscopy and its multi-photon variants have... 

Dilferently 2,6,8-trisubstituted 3-hydroxychromones such as 24 (Fig. 13) able to be readily uptaken by cells have been investigated by Dyrager et al for Live-Cell Imaging by multi-photon laser scanning microscopy (MPLSM). ... 

Optical clearing might be a fruitful mefliod for tissue spectroscopies (Raman, fluorescence, multi-photon, etc.), microscopies (confocal, OCT, laser scanning, near-field, etc), and imaging techniques (OCT, SHG, etc.), where scattering is a serious limitation. 

According to Eqs. (l)-(3),the generated SHG intensity depends on the square of the incident fight intensity, while the generated THG intensity will depend on the third power of the incident fight intensity. Similar to multi-photon induced fluorescence process, this nonlinear dependence allows localized excitation and is ideal for intrinsic optical sectioning in scanning laser microscopy. Usually the third-order nonlinear susceptibility is much weaker than the... 

In this chapter we describe advances in the femtosecond time-resolved multiphoton photoemission spectroscopy (TR-MPP) as a method for probing electronic structure and ultrafast interfacial charge transfer dynamics of adsorbate-covered solid surfaces. The focus is on surface science-based approaches that combine ultrafast optical pump probe excitation to induce nonlinear multi-photon photoemission (MPP) from clean or adsorbate covered single crystal surfaces. The photoemitted electrons transmit spectroscopic and dynamical information, which is captured by their energy analysis in real or reciprocal space. We examine how photoelectron spectroscopy and microscopy yield information on the unoccupied molecular structure, electron transfer and relaxation processes, light induced chemical and physical transformations and the evolution of coherent single particle and collective excitations at solid surfaces. 

In 2007, Beeby et al. nicely showed that two-photon time gated luminescence spectra of complexes could be measured using a cavity-dumped mode locked Ti-sapphire laser with two un-optimised Eu(III) complexes (complexes potential candidates for future time-gated two-photon microscopy studies. The next generation of functionalised Ln(III) complexes will permit the in cellulo study of biologically relevant analytes by two-photon luminescence microscopy. Multi-photon excitation microscopy has established itself as a powerfiil tool in the life sciences the... 

All those examples and some others [105] clearly illustrate the feasibility of multi-photon imaging microscopy using lanthanide complexes as probes. However, the very low nonlinear efficiency of these complexes is slightly compensated by the increase of the incident laser power intensity to obtain well resolved images, but this clearly remains an important drawback for any practical applications. 

Buist, A. H., Muller, M., Squier, J. and Brakenhofif, G. J. (1998). Real time two-photon absorption microscopy using multi point excitation. J. Microsc. 192, 217-26. 

The most frequently applied analytical methods used for characterizing bulk and layered systems (wafers and layers for microelectronics see the example in the schematic on the right-hand side) are summarized in Figure 9.4. Besides mass spectrometric techniques there are a multitude of alternative powerful analytical techniques for characterizing such multi-layered systems. The analytical methods used for determining trace and ultratrace elements in, for example, high purity materials for microelectronic applications include AAS (atomic absorption spectrometry), XRF (X-ray fluorescence analysis), ICP-OES (optical emission spectroscopy with inductively coupled plasma), NAA (neutron activation analysis) and others. For the characterization of layered systems or for the determination of surface contamination, XPS (X-ray photon electron spectroscopy), SEM-EDX (secondary electron microscopy combined with energy disperse X-ray analysis) and... 

Isobe, K, Watanabe, W., Matsunaga, S., Higashi, T., Fukui, K. and Itoh, K. (2005) Multi-spectral two-photon excited fluorescence microscopy using supercontinuum light source.Jpn. J. Appl. Phys., 44, L167-L169. 

Two-photon laser-scanning microscopy system. We use a Leica SP5 inverted 5 channel confocal microscope see Note 1 and Fig. la), a Ti Sapphire laser (Spectra Physics) with a 10-W pump tuned to 810 nm, a 20x multi-immersion objective with long working distance (for example, Leica 20x 0.7NA), an incubation cube chamber (Life Imaging Services, Basel, Switzerland) (rrrNote 1 and Fig. lb). 

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