Nonlinear microscopy

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... 

VIII. Multicontrast Nonlinear Microscopy of H E Stained Histological... 

This chapter introduces principles of three nonlinear contrast mechanisms— MPF, SHG, and THG—for microscopic imaging. The instrumentation of nonlinear multicontrast microscopy is described, and the methods of multicontrast image analysis are presented. The later parts of the chapter focus on several examples of multicontrast nonlinear microscopy applications in biological imaging. 

SCIA can be applied for correlation of three images obtained with multicontrast nonlinear microscopy. The two-image SCIA can be applied to correlate separately each two of the three images. However, direct correlation of all three images with SCIA algorithm renders quicker and more transparent results. To illustrate three-channel SCIA, an example is shown in Figure 4.2 where three channels (A, B, and C)... 

VIII. MULTICONTRAST NONLINEAR MICROSCOPY OF H E STAINED HISTOLOGICAL SECTIONS... 

Mertz, J. 2004. Nonlinear microscopy New techniques and applications. Review of overview of authors work in multiphoton microscopy. Curr Opin. Neumbiol. 14 610-16. 

Prent, N., Cisek, R., Greenhalgh, C., Sparrow, R., Rohitlall, N., Milkereit, M. S., Green, C., and Barzda, V. 2005b. Application of nonlinear microscopy for studying the structure and dynamics in biological systems. Proc. SPIE 5971 5971061-68. 

Squier, J. A., and Muller, M. 2001. High resolution nonlinear microscopy A review of sources and methods for achieving optimal imaging. Rev. Sci. Instrum. 72 2855-67. 

Q Imaging with Phase-Sensitive Narrowband Nonlinear Microscopy... 

Unlike in bulk nonlinear spectroscopy experiments, the signal in nonlinear microscopy is generated within a volume that is on the order of an optical wavelength. The axial extent of this volume is often referred to as the interaction length, which denotes the length within which the incident fields interact to produce a nonlinear polarization in the material. Such microscopic interaction lengths yield signal interference profiles that can differ markedly from those observed in macroscopic spectroscopy. 

In this chapter we explore several aspects of interferometric nonlinear microscopy. Our discussion is limited to methods that employ narrowband laser excitation i.e., interferences in the spectral domain are beyond the scope of this chapter. Phase-controlled spectral interferometry has been used extensively in broadband CARS microspectroscopy (Cui et al. 2006 Dudovich et al. 2002 Kee et al. 2006 Lim et al. 2005 Marks and Boppart 2004 Oron et al. 2003 Vacano et al. 2006), in addition to several applications in SHG (Tang et al. 2006) and two-photon excited fluorescence microscopy (Ando et al. 2002 Chuntonov et al. 2008 Dudovich et al. 2001 Tang et al. 2006). Here, we focus on interferences in the temporal and spatial domains for the purpose of generating new contrast mechanisms in the nonlinear imaging microscope. Special emphasis is given to the CARS technique, because it is sensitive to the phase response of the sample caused by the presence of spectroscopic resonances. 

We first review the essentials of the phase distribution of the electric fields at the focus of a high numerical aperture lens in Section II. After discussing the phase properties of the emitted signal, in Section HI we zoom in on how the information carried by the emitted held can be detected with phase-sensitive detection methods. Interferometric CARS imaging is presented as a useful technique for background suppression and signal enhancement. In Section IV, the principles of spatial interferometry in coherent microscopy are laid out and applications are discussed. The influence of phase distortions in turbid samples on phase-sensitive nonlinear microscopy is considered in Section V. Finally, in Section VI, we conclude this chapter with a brief discussion on the utility of phase-sensitive approaches to coherent microscopy. 

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