Multiphoton fluorescence

Detection of Higher Order Multiphoton Fluorescence from Organic Crystals... 

Multiphoton Fluorescence Imaging with the Near-Infrared 35 fs Laser Microscope... 

The NIR femtosecond laser microscope realized higher order multi photon excitation for aromatic compounds interferometric autocorrelation detection of the fluorescence from the microcrystals of the aromatic molecules confirmed that their excited states were produced not via stepwise multiphoton absorption but by simultaneous absorption of several photons. The microscope enabled us to obtain three-dimensional multiphoton fluorescence images with higher spatial resolution than that limited by the diffraction theory for one-photon excitation. 

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].

Xu C, Zipfel W, Shera JB, Williams RM, Webb WW (1996) Multiphoton fluorescence excitation new spectral window for biological nonlinear microscopy. Proc Natl Acad Sci USA 93 10763-10768... 

Written by an international panel of experts, this volume begins with a comparison of nonlinear optical spectroscopy and x-ray crystallography. The text examines the use of multiphoton fluorescence to study chemical phenomena in the skin, the use of nonlinear optics to enhance traditional optical spectroscopy, and the multimodal approach, which incorporates several spectroscopic techniques in one instrument. Later chapters explore Raman microscopy, third-harmonic generation microscopy, and nonlinear Raman microspectroscopy. The text explores the promise of beam shaping and the use of a broadband laser pulse generated through continuum generation and an optical pulse shaper. 

In Chapter 2, Hanson and Bardeen show a great use of multiphoton fluorescence to study chemical microenvironment in the skin. 

Ever since HRS has been developed as an experimental technique to determine the first hyperpolarizability p of molecules in solution, it has been realized that multiphoton fluorescence is a competing nonlinear process, contributing to the HRS signal [26]. For the classical dipolar and neutral molecules that may exhibit multiphoton fluorescence, electric-field-induced second-harmonic generation (EFISHG) experiments are possible. However, for ionic and non-dipolar compounds, no electric field can be applied over the solution. Hence, no EFISHG measurements are possible. Then it is very tempting to rely on the HRS measurement only. When there is, however, a multi-photon fluorescence (MPF) contribution, an overestimation of the first hyperpolarizability value results [27]. 

Larson DR, Zipfel WR, Williams RM, Clark SW, Bruchez MP, Wise FW, Webb WW (2003) Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 300 1434-1436... 

Xu, C., et al. Multiphoton fluorescence excitation new spectral windows for biological nonlinear microscopy. Proceedings of the National Academy of Sciences, USA, 1996, 93, 10763-10768. 

B. J. Bacskai, J. Skoch, G.A. Hickey, R. Allen, B.T. Hyman, Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques, J. Biomed. Opt 8, 368-375 (2003)... 

R.V. Krishnan, A. Masuda, V.E. Centonze, B. Herman, Quantitative imaging of protein-protein interactions by multiphoton fluorescence lifetime imaging microscopy using a streak camera, J. Biomed. Opt. 8, 362-267 (2003)... 

LT. Nieman, G.M. Krampert, and R. E. Martinez, An Apertureless Near-Field Scanning Optical Microscope and its Application to Surface-Enhanced Raman Spectroscopy and Multiphoton Fluorescence Imaging, Rev. Sci. Instrum. 72, 1691 (2001)... 

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