Recently, there has been great interest in a variety of novel ultrafast coherent anti-Stokes Raman scattering (CARS) methods that utilize broadband lasers (1–3), often involving narrowband probe pulses (4–6), chirped pulses (7,8), or fiber-broadened pulses (9–11) to obtain broadband vibrational information from molecules. With the variation of single-pulse, broadband, multiplex CARS microscopy discussed here, broad bandwidth spectra are acquired rapidly during the microscope scan and the spatially variant compositions of samples composed of multiple components are readily determined.

The essential feature of this method is the use of a single, ultra-broadband femtosecond (fs) pulsed-laser oscillator, which simultaneously provides all of the photons for the CARS scattering. By a combination of phase and polarization control of the 10-fs laser pulses, complete broadband spectral images are acquired in the vibrational fingerprint regime. An important advantage of this method is that full chemical composition analysis is obtained in a single microscope scan, rather than the multiple scans that are necessary with narrowband CARS microscopy (12–17). There are, however, tradeoffs necessary to gain this spectral width. With narrowband CARS, the signal sensitivity and signal acquisition rate can be much greater, but with broadband CARS, the spatial locations of multiple species can be obtained in a single scan. In the discussion that follows, the techniques of broadband CARS are discussed and illustrated with examples of multicomponent chemical imaging.

In general, various fluorescence microscopy techniques have become popular for highly sensitive imaging, but most methods require the introduction of fluorescent probes that can perturb the system of interest, and are often limited by photobleaching. On the other hand, infrared (IR) and Raman spectroscopy are excellent tools for identifying the chemical properties and physical environments of molecules within a condensed phase sample based upon intrinsic molecular vibrations. However, there are significant limitations to utilizing these techniques for microscopy. The long wavelengths required for IR absorption result in low spatial resolution, and spontaneous Raman scattering has a characteristically small cross-section. Also, sample fluorescence often overlaps the Stokes lines typically used in spontaneous Raman scattering spectroscopy. CARS has the potential to become a powerful spectroscopic tool due to the stronger signal levels of vibrational spectral information and three-dimensional sectioning capability (18).

The vibrational fingerprint region extends from about 800 cm^–1 to 1800 cm^–1 (19). Therefore, acquiring a full vibrational spectrum with ~1000 cm^–1 of bandwidth in this spectral region at each spatial location in a sample would enable discrimination of many sample components simultaneously, even those with similar spectral features (9). Furthermore, it is desirable to obtain this full spectrum in single-pulse mode, without scanning over each frequency region individually.

With the goal of imaging complex samples in mind, several groups have developed highly refined methods to increase the sensitivity of broadband CARS spectroscopy to combine it with microscopy as a label-free, chemically selective imaging technique (1–4,6–11). However, due to the time it typically takes to collect the full spectrum, to our knowledge, only a few groups have constructed microscope images with a CARS spectrum at each pixel (6,9,11).

Many applications would benefit from a sensitive imaging technique that provides chemical selectivity for multiple components simultaneously. For example, it would be valuable to obtain the spatial distribution of specific molecules throughout living biological cells and tissues. In particular, the segregation of lipids into distinct domains within the outermost cell membrane is an area of active investigation with implications for an understanding of protein sorting, cell–cell interactions, and signaling processes (20). Recently, narrowband and multiplex CARS microscopy has been used to image the distribution of two different lipids in model lipid membranes by tuning to a single vibrational band or spectral region of interest (6,14). By increasing the spectral bandwidth, it may be possible to determine the precise chemical composition of such domains without extrinsic chemical markers, which are usually necessary to discriminate between different species in a sample. Recent work in narrowband CARS imaging of living cells (11,16,17) makes the prospect of sensitive broadband CARS for imaging biological systems also potentially exciting.