The development opened of novel imaging technologies has new doors in our understanding of the nervous system in health and disease. For instance, electron microscopy (EM) has revealed the structure of the myelin sheath with unparalleled resolution [1
]. Magnetic resonance imaging allows for the noninvasive study of lesion progression in neurological diseases such as multiple sclerosis [2
] and confocal microscopy studies have revealed the dynamics of microglial cell activation in response to trauma in rat brain slices [3
]. Despite the advances facilitated by these technologies, the limitations posed by them highlight the need for a more robust imaging method. Dehydration and staining in EM preclude its application for observing real-time processes. Magnetic resonance imaging lacks single-cell resolution. Confocal fluorescence is hindered both by low penetration depth limiting its applications for in vivo
studies and by photobleaching that can complicate image acquisition and analysis.
Nonlinear optical (NLO) microscopy [4
] is becoming a powerful tool for studying live tissues and live animals due to several unique advantages over traditional methods. Nonlinear dependence on excitation intensity gives NLO microscopy inherent 3-D imaging capability without the need for a confocal pinhole. This is particularly advantageous in the case of tissue and in vivo
imaging where significant scattering can reduce the signal collection efficiency by confocal detection. Laser scanning facilitates real-time imaging of live tissues and animals [5
]. Also, NLO microscopy utilizes near-IR excitation that provides both superior optical penetration into tissues [6
] as well as reduced photodamage due to reduced interaction with endogenous molecules [7
]. Furthermore, different NLO imaging modalities are sensitive for probing different cellular structures.
In order to provide a robust imaging system, it is necessary to combine several NLO imaging modalities on the same platform. Multimodality in NLO imaging has been demonstrated to be a valuable tool for bioimaging. Fu et al.
demonstrated simultaneous coherent anti-Stokes Raman scattering (CARS) imaging of axonal myelin and sum-frequency generation (SFG) imaging of astrocyte processes in live spinal tissues [8
]. Nan et al.
combined CARS with two-photon-excited fluorescence (TPEF) to investigate the relationship between mitochondria and lipid droplets in tumor cells [9
]. Multimodality was also utilized to investigate the impact of obesity on tumor stroma [10
] as well as to map atheroma in pig artery based on molecular composition [11
In this paper, we systematically describe the integration of CARS, SFG, and TPEF microscopy. We discuss their unique advantages, the parameters to be considered in constructing a multimodal NLO microscope, and how to realize such a system. Finally, we show biomedical applications in that multimodal NLO microscopy was utilized to reveal the relationship between different structures in central nervous system and calcium ion influx into axons during demyelination, demonstrating its potential in addressing biological and biomedical questions.