Multimodal Nonlinear Optical Microscopy and Applications to Central Nervous System Imaging

Multimodal nonlinear optical (NLO) imaging is poised to become a powerful tool in bioimaging given its ability to capitalize on the unique advantages possessed by different NLO imaging modalities. The integration of different imaging modalities such as two-photon-excited fluorescence, sum frequency generation, and coherent anti-Stokes Raman scattering on the same platform can facilitate simultaneous imaging of different biological structures. Parameters to be considered in constructing a multimodal NLO microscope are discussed with emphasis on achieving a compromise in these parameters for efficient signal generation with each imaging modality. As an example of biomedical applications, multimodal NLO imaging is utilized to investigate the central nervous system in healthy and diseased states.

I. Introduction

The development of novel imaging technologies has opened 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.