Coherent AntiStokes Raman Scattering CARS Microscopy
Coherent anti-Stokes Raman scattering (abbreviated as CARS) is a third-order nonlinear optical process that can produce a vibrational transition. This nonlinear optical process has been discussed in Chapter 5. For CARS, two optical beams of frequencies vp and vs interact in the sample to generate an anti-Stokes optical output at vAS = 2vp - vs in the phase-matched direction (a specific direction). The signal has an electronic contribution (from the electronic third-order nonlinear optical response), but is resonantly enhanced if vp - vs coincides with the frequency of a Raman active molecular vibration (see Chapter 4). The molecular vibration involved in a CARS signal enhancement can then be used as a contrast mechanism for bioimaging. Since the first attempt of Duncan et al. (1982), CARS microscopy has attracted a great deal of attention in recent years (Zumbusch et al., 1999; Muller et al., 2000; Potma et al., 2000; Hashimoto et al., 2000; Cheng et al., 2001;Volker et al., 2002). Xie
and co-workers have made significant advances in the application of the laser scanning CARS microscopy to cell biology (Zumbusch et al., 1999; Cheng et al., 2001; Volkmer et al., 2001). The CARS microscopy provides a number of advantages, some of which are (Volker et al., 2001):
• Vibrational contrast in the CARS microscopy is inherent to the cellular species, thus requiring no endogenous or exogenous fluorophores that may be prone to photobleaching.
• CARS, being a coherent optical process (phase matching), offers much higher sensitivity than the spontaneous Raman process.
• CARS, being a nonlinear optical process, exhibits a nonlinear dependence on the pump intensity and, like in two-photon or other nonlinear microscopy described above, generates signals from the focal volume. This feature allows three-dimensional optical sectioning of thick samples to obtain a high-resolution three-dimensional image.
• CARS can provide chemical selectivity as different vibrational modes can be used for contrast.
• The CARS signal can be detected even in the presence of background autofluorescence, since a CARS signal is highly directional because of the phase-matching requirement.
CARS imaging is complicated by background signals derived from two sources: (i) the nonresonant electronic contributions that exist even when the Raman resonance condition is not met and (ii) electronic and Raman contributions from the solvent. The latter is particularly troublesome when using an aqueous medium as water generates a strong resonant CARS signal. Volker et al. (2001) have shown that by detecting CARS signal in the backward direction (which they call E-CARS) one can effectively suppress the solvent background and significantly increase the sensitivity of the CARS microscopy.
High-peak power lasers are needed to enhance the nonlinear optical process of CARS. At the same time it is necessary to maintain a narrow bandwidth of pulses in order to obtain good spectral resolution for selectively using a specific vibrational mode for resonance enhancement. Cheng et al. (2001) suggested the use of picosecond pulses instead of femtosecond pulses because the former allows one to achieve a better signal-to-background ratio (Cheng et al., 2001). Furthermore, to obtain and maintain overlap of two femtosecond laser pulses is much more difficult, in comparison with overlap to obtain and maintain of two picosecond pulses. The schematics of their experimental arrangement is shown in Figure 7.24. It utilizes two synchronized mode-locked Ti: sapphire lasers producing picosecond pulses at the 80-MHz repetition rate. The pump beam with frequency vp is tunable from 690 to 840 nm, while the Stokes beam with frequency vs is tunable from 770 to 900 nm. This arrangement allows one to cover the vibrational frequency range from 100 to 3400 cm-1. They used this arrangement to obtain the CARS image of unstained human epithelial cells. The Raman band at 1570 cm-1, arising from proteins and nucleic acid, was used.
FUTURE DIRECTIONS OF OPTICAL BIOIMAGING
FUTURE DIRECTIONS OF OPTICAL BIOIMAGING
- Figure 7.24. Schematics of a synchronized mode-locked picosecond Ti:sapphire laser system for backward detection CARS microscopy. Millenia is the diode-pumped Nd laser. Tsunami is the Ti:sapphire laser. (Reproduced with permission from Cheng, et al., 2001.)
Post a comment