ABSTRACT
I. Introduction: Nonlinear Spectroscopic Signals for Microscopic Imaging ... 168 II. Broadband Femtosecond Pulses in Microscopy ........................................... 171 III. Experimental Implementation: Sources and Pulse Shapers ......................... 174 IV. In Situ Pulse Compression and Characterization .......................................... 177 V. Single-Beam CARS Microspectroscopy and Microscopy ........................... 179
A. Introduction to Femtosecond and Single-Beam CARS ........................ 179 B. Single-Beam Time-Resolved CARS Microspectroscopy ..................... 184 C. Time-Domain Control and Observation of Molecular
Vibrations in CARS Microspectroscopy .............................................. 185 D. Application to White Powder Identification and Threat Assessment ... 186 E. Chemical Imaging ................................................................................. 186 F. Improving Sensitivity with Heterodyne CARS Detection .................... 188
VI. Broadband Multiphoton Fluorescence Microspectroscopy .......................... 190 VII. Conclusions and Outlook .............................................................................. 192 Acknowledgments .................................................................................................. 192 References .............................................................................................................. 192
In recent years, multiphoton microscopy has emerged as a new tool for bio-imaging, offering an unprecedented wealth of information (Zipfel et al. 2003; Xie et al. 2006). This includes fluorescence imaging with highly reduced photobleaching of the labeling dyes, bright images with very high three-dimensional resolution, deep-tissue imaging, and novel contrast mechanisms. In the most commonly employed two-photon fluorescence (TPF) microscopy, labeling fluorophores are excited simultaneously with two photons, which can roughly be understood as being equivalent to an excitation with a single photon of double energy, or half the wavelength (Figure 7.1a). The use of long excitation wavelengths in the near-infrared spectral region is the reason why multiphoton techniques usually achieve much higher penetration depths, as scattering is highly reduced. Also, the involvement of two photons in the excitation leads to a quadratic intensity dependence of the fluorescence. On the one hand, this requires pulsed laser sources for efficient excitation with high peak intensities but, on the other hand, it confines the signal generation to the focal volume, which is the reason for the highly beneficial 3D-imaging capabilities. Therefore, it is clear that at a given average laser power the shortest possible pulses achieve the highest multiphoton signal levels.
