In the past 30 years major advances in medical imaging have been made in areas such as magnetic resonance
imaging, computed tomography, and ultrasound. These techniques have become quite effective for structural
imaging at the organ or tissue level, but do not address the clear need for imaging technologies that exploit
existing knowledge of the genetic and molecular bases of disease. Techniques that can provide similar
information on the cellular and molecular scale would be very powerful, and ultimately the extension of such
techniques to in vivo measurements will be desired. The availability of these imaging capabilities would allow
monitoring of the early stages of disease or therapy, for example.
Optical techniques provide excellent imaging capabilities, with
sub-micron spatial resolution, and are noninvasive.
An overall goal of biomedical imaging is to obtain diagnostic or functional information about
biological structures. The difficulty of acquiring high-resolution images of structures deep in tissue presents a
major challenge, however, owing to strong scattering of light. As a consequence, optical imaging has been
limited to thin (typically ~0.5 mm) samples or superficial tissue. In contrast, techniques such as ultrasound and
magnetic resonance provide images of structures centimeters deep in tissue, with ~100-micron resolution. It is
desirable to develop techniques that offer the resolution of optics with the depth-penetration of other techniques.
Since 1990, a variety of nonlinear microscopies have been demonstrated. These include 2- and 3-photon
fluorescence microscopy, and 2nd- and 3rd-harmonic generation microscopies. These typically employ
femtosecond-pulse excitation, for maximum peak power (and thus nonlinear excitation) for a given pulse
energy. A relative newcomer to the group is CARS microscopy , which exploits resonant vibrational
excitation of molecules or bonds. The CARS signal contrast arises from intrinsic elements of cells, and thus
CARS offers the major advantages of a label-free technique. In contrast to other nonlinear microscopies, CARS
imaging is best performed with excitation pulses in the 2-7 ps range, which overlap spectrally with the desired
Raman resonances. Two synchronized excitation pulses are required at different wavelengths, and these beat to
excite the vibration.