Microscopy nonlinear contrast mechanisms

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. 

Nonlinear light-matter interactions have been successfully applied to create new visualization contrast mechanisms for optical microscopy. Nonlinear optical microscopy employs femtosecond and picosecond lasers to achieve a high photon flux density by focusing the beam onto a sample with a high numerical aperture (NA) microscope... 

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. 

The complex quantity, y6br = e (y(3)r) + i Im (x r), represents the nuclear response of the molecules. The induced polarization is resonantly enhanced when the Raman shift wp — ws matches the frequency Qr of a Raman-active molecular vibration (Fig. 6.1A). Therefore, y(3)r provides the intrinsic vibrational contrast mechanism in CRS-based microscopies. The nonresonant term y6bnr represents the electronic response of both the one-photon and the two-photon electronic transitions [30]. Typically, near-infrared laser pulses are used to prevent the effect of two-photon electronic resonances. With input laser pulse frequencies away from electronic resonances, y(3)nr is independent of frequency and is a real quantity. It is important to realize that the nonresonant contribution to the total nonlinear polarization is simply a source for an unspecific background signal, which provides no chemical contrast in some of the CRS microscopies. While CARS detection can be significantly effected by the nonresonant contribution y6bnr [30], SRS detection is inherently insensitive to it [27, 29]. As will be discussed in detail in Sects. 6.3 and 6.4, this has major consequences for the image contrast mechanism of CARS and SRS microscopy, respectively. 

The chemical specificity of CARS microscopy is readily combined with other nonlinear optical image contrast mechanisms, such as two-photon fluorescence (TPF), SHG, and THG, resulting in a multimodal CARS microscopy [88, 118, 117, 43]. In multimodal nonlinear optical imaging, TPF, SHG, and THG signals all benefit from the use of femtosecond laser pulses of high peak intensities, whereas the contrast and chemical selectivity of CARS benefits from the use of picosecond (narrow-bandwidth) pulses (see discussion in Sect. 6.2.3). As demonstrated by Pegoraro et al. [43], this apparent... 

---