Coherent Anti-Stokes Raman Scattering Microscopy

Breakthroughs in biology often arise from the development of new microscopy tools. Our understanding of biological systems has greatly benefited from the studies with electron microscopy, phase contrast microscopy, confocal fluorescence microscopy, scanning probe microscopy, and other imaging tools. In recent years, the coupling of nonlinear optics with scanning microscopy opened up a new window through which to watch the biological world. The tight focusing condition in microscopy allows an efficient generation of nonlinear optical (NLO) signals even with low pulse energies. Meanwhile, the nonlinear dependence on excitation intensity ensures that the NLO signal is only generated in the focal center, providing an inherent three-dimensional (3D) spatial resolution. In this sense, nonlinear optics was “born” for imaging use. The earliest development of NLO microscopy can be traced back to the 1970s when second-harmonic imaging of crystals was reported as a proof-of-principle of second-harmonic generation (SHG) microscopy.1,2 In 1982, Duncan and co-workers reported coherent anti-Stokes Raman scattering (CARS) imaging of D2O in onion cells using a dye laser system.3 However, these early papers did not prompt a lot activity in the 1980s. The prosperity of the multiphoton and NLO microscopy field was triggered by the demonstration of two-photon excitation fluorescence (TPEF) imaging with a mode-locked femtosecond dye laser in 1990,4 followed by demonstrations of high-quality third harmonic generation (THG) imaging5 and CARS imaging6 in the late 1990s. Nowadays, TPEF and SHG microscopy have become powerful tools for biological research.7,8 The first systematic study of the CARS phenomenon was published in 1965 by two scientists from the Ford Motor company.9 CARS spectroscopy emerged in the early 1970s10 and has been one of the most important coherent Raman spectroscopy techniques.11–13 In a CARS process, three laser fields at the pump (ωp), Stokes (ωs), and probe ( ωp′) frequencies interact with a medium to generate a new field at the anti-Stokes frequency ωas=(ωp−ωs)+ωp′. In most experiments, the pump field Ep and probe field Ep′ come from the same laser beam. The CARS signal arises from the third-order-induced polarization, P(3)=χ(3)EpEs∗Ep. CARS occurs in any medium with nonzero susceptibility χ(3). Importantly, the CARS signal can be significantly enhanced when the beating frequency, (ωp − ωs), is in resonance with a molecular vibration (Fig. 1A). By scanning (ωp − ωs), one can acquire a CARS spectrum that carries essentially the same information as the spontaneous Raman spectrum of the same sample. However, because of the interference between the Raman component and the nonresonant component, the CARS line profile is dispersed, with a shift of the peak to lower frequency and appearance of a dip at higher frequency (Fig. 1B). Fig. 1 (A) Energy diagram of CARS. (B) CARS and Raman spectra of polystyrene in the 1550 to 1650 cm−1 region. The nonresonant background is indicated by the dashed line. The red shift of the CARS peaks and the dip at 1608 cm−1 result from the ... In a CARS experiment, other coherent Raman processes occur simultaneously, including coherent Stokes Raman scattering (CSRS), stimulated Raman gain (SRG), stimulated Raman loss (SRL), and Raman-induced Kerr effect (RIKE).11,14 In CSRS, a new field at frequency ωs − (ωp − ωs) is generated. In SRG, the ωs field experiences a gain while in SRL the ωp field experiences a loss. In RIKE, new polarization components of the excitation fields are generated. In most CARS imaging experiments, two parallel-polarized beams are used, where RIKE is negligible (χ2111 = 0 for an isotropic medium). In contrast to SRG, SRL, and RIKE, the CARS signal is spectrally separated from the excitation beams, making it easy to filter out the linear scattering from a heterogeneous biological system. Compared with CSRS, the CARS signal appears at a wavelength shorter than excitation wavelengths, usually in the visible region where detectors of high quantum efficiency are available. In CARS microscopy,15–18 the pump and Stokes beams are tightly focused into a sample and a CARS image is generated by scanning either the sample or the laser beams. Besides the 3D sectioning capability, CARS microscopy offers several unique advantages. CARS microscopy permits nondestructive molecular imaging without any labeling. This advantage is important for imaging small molecules such as lipids or drugs for which the labeling may significantly affect the molecular properties. The coherent addition of CARS fields results in a quadratic signal increase with respect to the number of molecular oscillators, versus the linear increase of spontaneous Raman signal. The coherent addition also results in a highly directional output, which greatly facilitates the signal collection. The large CARS signal allows high-speed vibrational imaging, which is not possible with Raman microscopy. CARS signals appear at a wavelength that is shorter than excitation wavelengths, spectrally separated from the one-photon fluorescence background. The major disadvantage of CARS is the existence of a nonresonant background which arises from the electronic contribution to χ (3). This nonresonant background is coherently mixed with the vibrationally resonant signal, which limits the sensitivity of CARS in detecting weak Raman bands. Additionally, the resonant CARS signal decreases quadratically with the molecular concentration, making the detection of low-concentration molecules difficult. In the past few years, tremendous efforts have been spent on suppressing the nonresonant background and pushing the detection sensitivity limit. This paper aims to evaluate the potential and limitations of CARS microscopy through a review of recent advances. The rest of the paper will discuss the technical developments, summarize the biological and biomedical applications, and finally present an outlook.