The phenomenon of stimulated Raman scattering (SRS) was discovered immediately after the laser was invented (58
). When a cell filled with nitrobenzene was introduced into a ruby laser cavity, Woodbury and Ng observed a rather strong emission at a new wavelength other than the fundamental wavelength of ruby laser, which was later understood as stimulated Raman gain (58
). Two years later, a related phenomenon, stimulated Raman loss (or inverse Raman) was also discovered (59
). Since then stimulated Raman spectroscopy has been performed on various physical and chemical systems (61
). In particular, femtosecond stimulated Raman spectroscopy has been developed to provide vibrational structural information with both high temporal and spectral information of chromophore systems such as primary photoisomerization and green fluorescent protein (64
SRS probes the excited vibrational population instead of the vibrational coherence detected by CARS (11
). When Ω is tuned into a vibrational resonance, Ω→ωv
, due to the combined interaction of the incident pump and probe beams, the rate of the vibrational excitation will be greatly accelerated compared to that in spontaneous Raman scattering by a factor given by
is the (normally large) number of photons in the optical mode of the probe beam (25
). Such efficient excitation of a molecular vibrational level obviously requires energy input from the laser fields. As required by energy conservation, for each quantum of the vibrational excitation being excited, it is accompanied by one photon being annihilated from the pump beam and simultaneously a photon being created into the probe beam (). The resulting intensity loss in the pump beam is called stimulated Raman loss, and the intensity gain in the probe beam is called stimulated Raman gain.
Figure 4 Principle of stimulated Raman scattering microscopy. (a) Energy diagram of stimulated Raman scattering when the energy difference between pump and probe is resonant with a vibrational transition. Stimulated Raman gain of the probe beam and stimulated (more ...)
Stimulated Raman gain and loss can also be understood in the semi-classical framework of nonlinear induced polarization (60
) as an optical heterodyne phenomenon. When Ω→ωv
, besides the CARS radiation at the anti-Stokes frequency, two other third-order induced polarizations, ppump
, are generated at the fundamental pump and probe frequencies, shown in , respectively:
propagate in the forward direction and interfere with the incident pump and probes fields with their corresponding phases. For stimulated Raman gain, pprobe
constructively interferes with Eprobe
and results in an intensity gain:
For stimulated Raman loss, ppump
destructively interferes with Epump
and results in an intensity loss:
Such an optical heterodyne interpretation is analogous to the picture that linear absorption can be treated as the destructive interference between the incident field and linear induced polarization of the molecule at the forward detector.
SRS as a contrast mechanism for microscopy was first reported using multiplex detection with a photodiode array in combination with a femtosecond amplified laser system (66
). Although the amplified laser system generates a large SRS signal, it is not suitable for bio-imaging because the excessive peak power causes sample damage and the low repetition rate limits the image acquisition speed. Instead, using narrow-band picosecond pulse trains with high repetition rates, stimulated Raman scattering was later adapted into a high-frequency modulation transfer microscopy by several groups (67
). Very recently, its multiplex version has been developed into a spectral imaging modality by using a spectrally shaped broadband excitation pulse (70
). When the pump beam is blocked, the probe beam maintains its intensity after passing through the sample; when the pump beam is unblocked, the probe beam experiences stimulated Raman gain due to nonlinear interactions. Hence, a temporal modulation of the pump beam intensity at a frequency f
would give rise to a modulation of the probe beam intensity, at the same frequency f
, after interacting with the vibrational oscillators at the foci.
SRS imaging is free from the non-resonant background in CARS microscopy. shows the simultaneous SRS CH2 image of the same worm sample in . Only purely lipid contrast is visible in SRS. This is so because, in the absence of a vibrational eigenstate that could hold the population and energy, energy simply cannot transfer from the pump beam to the probe beam, as required by energy conservation. In the optical heterodyne picture, the off-resonant polarization fields are either 90 degree ahead or 90 degree behind the incident pump or probe fields at the detector, which forbids any constructive or destructive interference (and hence intensity gain or loss) with the pump or probe beams from occurring.
Such a drastic contrast between SRS and CARS is analogous to the more familiar relation between absorption and Rayleigh scattering. While linear absorption by a molecule can be tuned off completely from its absorption band, Rayleigh scattering always occurs even if there is no resonance between the light and the molecule. Physically, scattering events can be mediated by a virtual state, while absorption events cannot. To some extent, SRS and CARS can be viewed as the nonlinear Raman analog of the linear absorption and Rayleigh scattering phenomena, respectively.
SRS overcomes all major difficulties associated with CARS microscopy, as summarized in . First, the absence of the non-resonant background eliminates the biggest obstacle for CARS imaging quantification and interpretation. Second, without the interference effect from the background, the SRS spectrum is identical to that of spontaneous Raman scattering (), allowing the straightforward utilization of all the accumulated knowledge of Raman spectroscopy. Third, the detection sensitivity of SRS is demonstrated to be much higher than that of CARS microscopy. The signal-to-noise ratio of SRS detection may be written
denotes the laser intensity noise of the probe beam, and
is the shot noise of the probe beam intensity. Thanks to the high-frequency modulation and lock-in detection at a high f
can be readily removed in SRS detection. With α
→ 0, SRS can reach the shot noise limit, with detectable ΔIp
within one second of acquisition time.
Comparison of spectroscopy and microsocpy aspects between CARS and SRS imaging.
It is worth noting that, based on Eqs. (3
) and (6
in the scenario in which laser intensity fluctuation can be completely eliminated and the shot noise (from non-resonant background and the probe beam for CARS and SRS, respectively) is the only remaining noise source. However, it is extremely hard for CARS to meet this ideal situation, because of the difficulty of employing an effective high frequency modulation technique. In CARS, when certain optical properties (e.g., frequency) of the pump or probe beam are modulated, the non-resonant background almost always leaves spurious intensity noise. In contrast, the probe beam in SRS is unperturbed before interacting with the sample, and its intensity noise can be circumvented with ease by modulation transfer.
Moreover, SRS exhibits a few other favorable properties over CARS (). The concentration dependence of CARS turns over from a quadratic in high concentration limit to a linear in the limit of low analyte concentration (Eq. (2)
), with the exact quantitative relation depending on the nonlinear nature of the surrounding solvent. In contrast, the strict linear concentration dependence of SRS permits straightforward and reliable quantification. In addition, because SRS involves measurements of transmission differences of the input beams, SRS is automatically phase matched. Hence, there exists a well-defined point spread function that can be used for image deconvolution (). Therefore, the image contrast in SRS microscopy is easy to understand, because it is free from spatial coherence artifacts.
Although the phase matching condition dictates that the SRS effect be detected by measuring the transmitted pump or Stoke beams in the forward direction, it is desirable to detect SRS in the backward direction for thick, non-transparent tissue samples as light does not penetrate through them. Fortunately, this can be done if a large area detector is used to collect a significant portion of the back-scattered light after the SRS signal is already generated at the laser focus (71
Compared to spontaneous Raman microscopy, stimulated Raman scattering exhibits an orders-of-magnitude faster imaging speed by virtue of optical amplification of the vibrational excitation rate. Photon energy dissipates into vibrational levels during both Raman processes, but with drastically different efficiency. As shown by Eq. (4)
, the acceleration factor, rstim. Raman
, could be estimated for the SRS imaging apparatus reported in Ref. (67
). 5mM methanol, which corresponds to about 300,000 C-H vibrational oscillators within the laser focal volume, gives a stimulated Raman loss signal of about ΔISRS
. With a known σRaman
for one C-H bond, the total spontaneous Raman scattering cross sections of 3*105
C-H vibrational oscillators will add up to be 3*10−24
. Given the laser waist area of pump beam being 10−9
under a tight focus, one would expect to produce a relative spontaneous Raman scattering signal with ΔIspon.Raman
) ~ 3*10−15
. Therefore, rstim.Raman
is estimated to be as high as 7*10−8
, which accounts for the orders-of-magnitude acceleration of imaging speed so that video-rate SRS microscopy for live animal imaging becomes feasible (71
Having achieved label-free vibrational specificity, unprecedented imaging speed and superb detection sensitivity, SRS has opened up a wide range of chemical imaging applications in biomedical science and technology by targeting various vibrational bands (see ). As shown in , live cells can be imaged without external labeling by directly targeting different chemical moieties (67
). Tissue pathologies (72
) and food products (73
) can be analyzed without applying any dye staining. Reaction kinetics of biopolymer lignin under a chemical treatment can be imaged in situ
with high spatial and temporal resolution (74
). Small molecules such as drugs and metabolites can be monitored and followed inside tissues, as shown in . Lipid storage of C. elegans
and its genetic regulation can be explored in vivo
when combined with genetic manipulation of this model organism (75
). As illustrated in , unlike CARS microscopy, SRS only probes the lipid contribution from intestine and hypodermal without the non-resonant background contribution from other tissues, representing a major advantage for high-throughput genetic screening analysis. By implementing a multiplex spectral imaging mode with a spectrally shaped broadband excitation pulse, more specific and detailed spectral features in the congested C-H and O-H region (2800~3100cm−1
) can be efficiently picked up even in the presence of interfering species (70
Vibrational bands and corresponding Raman shifts used in SRS microscopy
Figure 5 SRS imaging of live cells at various spectral regions. (a) SRS image and (b) optical transmission microscope image of an unstained tobacco BY2 cultured cell with the Raman shift being set to 2967 cm−1. The nucleus and cell walls of a tobacco BY2 (more ...)
Figure 6 Tissue imaging by SRS microscopy. Distributions of (a) topically applied compound retinoic acid and (b) penetration enhancer dimethyl sulfoxide (DMSO) in a mouse ear skin. These images were acquired when tuned into the Raman shifts (c) of retinoic acid (more ...)
Another nonlinear dissipation coherent process is two-photon absorption. Historically two-photon absorption was the first nonlinear quantum transition to be explored, having been predicted in 1931 by Goeppert-Mayer (76
). The widely used two-photon excited fluorescence spectroscopy and microscopy (7
) are based on the sensitive detection of the subsequent fluorescence emission following two-photon absorption by fluorophores. Two-photon absorption is a nearly simultaneous absorption of two low-energy photons in order to excite a molecule from one state (usually the ground state) to a higher energy electronic state. The sum of the energies of the two photons is resonant with the energy difference between the lower and upper states of the molecule. It fundamentally differs from linear optical absorption in that the strength of absorption depends on the square of the light intensity, and the quantuam mechancal selection rules are different.
Normally two-photon absorption as in two-photon excited fluorescence microscopy (7
) is operated under a single beam mode in which molecules are excited by an ultrafast (normally femtosecond or picosecond) pulse train from a mode-locked laser such as a Titanium-Sapphire laser. The two photons involved are drawn from the same laser beam, and thus have similar frequencies within the laser pulse bandwidth. Hence it is difficult to distinguish these two photons spectrally with such a single beam mode.
Two-color dual beam excitation scheme permits two-photon absorption to be compatible with modulation transfer microscopy (77
). In principle, two-photon absorption can be equally induced by two photons with different colors, as long as the sum of the energies of the two photons again matches the targeted electronic transition, the two laser pulse trains are temporally synchronized and t overlapped in space. In such a dual-beam mode, blocking the intensity of either color terminates the absorption of the other color by the molecules, as the successful absorption event necessitates the simultaneous presence of the two beams. Two-photon absorption microscopy provides contrast mechanisms for non-fluoresscent chromophores that have appreciable two-photon absorption cross sections (77
), as shown in . In the area of biomedicine, examples include beta-carotene, oxy-hemoglobin, deoxy-hemoglobin, melanin and cytochromes.
Figure 7 Two-photon absorption microscopy. (a) Energy diagram of simultaneous two-photon absorption by a high-lying electronic state through an intermediate virtual state. (b) 3D volume rendering of two-photon absorption signal from human melanoma lesions obtained (more ...)
Dual-beam two photon absorption microscopy and stimulated Raman scattering microscopy are spectroscopically related to each other. They both operate through simutaneous two photon transitions, with one photon drawn from the pump beam and one drawn from the probe beam, respectively, mediated through a virtual state. The difference is that, in the former, the probe photon continues to excite the molecule up to higher energy levels, while in the latter the probe photon brings the molecule down to the vibrational excited state in the ground electronic manifold. In addition, both the response functions of two photon absorption microscopy and stimulated Raman scattering microscopy for a given molcule are given by the imaginary part of third-order nonlinear susceptibility (25
). The difference lies in the fact that the former corresponds to two-photon resonance while the latter is assocated with the vibrational resonance.