Optical microscopy is unique among current imaging modalities in its ability to probe living specimens with subcellular resolution, enabling the visualization of morphological details in tissue and cells on the scale of a few hundred nanometers. The discovery and broad application of genetically encoded fluorophores, such as GFP and YFP, to tag specific proteins has had a considerable impact on modern microscopy. Additionally, fluorescence microscopy has superb sensitivity (see single molecule section). Labeling and staining may, however, perturb the studied system. This is especially true for small molecules, such as metabolites and drugs, for which the attachment of ‘big’ labels can change the biological function. Coherent Raman imaging provides a means for label-free imaging of biological specimens and tissues.

Vibrational spectra of biological specimens contain a multitude of molecular signatures that can be used for identifying biochemical constituents in tissue. Infrared absorption (FT-IR) microscopy is limited by low spatial resolution due to the long wavelength of infrared light and low penetration depth into biological specimens due to strong water absorption. Spontaneous Raman scattering [Fig.1a], overcoming the problems associated with the long excitation wavelengths, lacks the sensitivity required for rapid imaging. Stimulated Raman Scattering (SRS) and Coherent anti-Stokes Raman Scattering (CARS), shown in Fig.1b and 1c, respectively, allow the enhancement of the weak Raman signal by means of nonlinear excitation, enabling imaging to speed up to video rate.

Fig.2: Input and output spectra of SRS and CARS:

SRS leads to an intensity increase in the Stokes beam (SRG) and an intensity decrease in the pump beam (SRL). Also shown (not to scale) is the CARS signal generated at the anti-Stokes frequency.

Stimulated Raman Scattering (SRS) Microscopy

In spontaneous Raman scattering, only one laser beam at a frequency ωp illuminates the sample and the signal is generated at the Stokes and anti-Stokes frequencies, ωs and ωas, respectively, due to inelastic scattering. In SRS, however, two laser beams at ωp and ωS coincide on the sample.  When the difference frequency Δω=ωp-ωS (also called the Raman shift), matches a particular molecular vibrational frequency Ω, amplification of the Raman signal is achieved by virtue of stimulated excitation of molecular transition rate r.

                r ~ σRaman· np· (nS +1)    

                                      
σRaman is the (Raman-shift dependent) Raman scattering cross-section of the molecule and np and nS are the number of photons per mode in the pump and Stokes fields, respectively. In the absence of the Stokes-beam (nS=0), the unity in the equation above accounts for spontaneous Raman scattering. Under our typical excitation condition nS is, however,  bigger than 10^7; hence, stimulated Raman scattering provides amplification at the vibrational transition rate. As a consequence of the amplified energy transition rate, the intensity of the Stokes beam, IS, experiences a gain, ΔIS, (stimulated Raman gain, SRG) and the intensity of the pump beam, Ip, experiences a loss, ΔIp, (stimulated Raman loss, SRL).  Either ΔIS  or ΔIp can be used as vibrational contrast for SRG and SRL microscopy, respectively.

SRS cannot occur when Δω does not match any vibrational resonance that absorbs the difference energy from the fields. Thus, SRS does not have a nonresonant background signal.  The intensity of SRG or SRL is described by  ΔIS ~ N · σRaman· Ip· IS  and  ΔIp ~ - N · σRaman· Ip· IS , where N is the number of molecules in the probe volume. As in two-photon microscopy, the nonlinearity of SRS in the overall excitation intensity is the basis for 3D sectioning,  which allows for point-by-point three-dimensional imaging of thick specimens.

Coherent anti-Stokes Raman scatttering (CARS) Microscopy

CARS is a four-wave mixing process in which a pump beam at frequency ωp and a Stokes beam at frequency ωs interact with a sample to generate an anti-Stokes signal at frequency ωas=2ωp - ωS . When the beat frequency between the pump and Stokes Δω matches the frequency of a particular Raman active molecular vibration Ω, the resonant oscillators are coherently driven. This generates a nonlinear polarization of the sample that radiates the anti-Stokes signal. Because the radiation is emitted coherently, the CARS signal is strongly enhanced compared to the spontaneous Raman scattering signal, for which Stokes and anti-Stokes photons are emitted incoherently.

In contrast to SRS, CARS is a parametric process, i.e., no energy is transferred into the sample but the difference energy between the pump- and Stokes-photon is carried away by the anti-Stokes field.  As a result, CARS can also occur when there are no resonant molecules in the focal volumes. This gives rise to a nonresonant background that can limit the sensitivity and alter the CARS spectra from spontaneous Raman spectra. We developed several detection schemes for CARS microscopy to overcome this background.

Fig.3: SRS tissue imaging of fresh mouse skin. For the acquisition of this image stack in mouse ear, SRL image contrast was tuned into the CH2-stretching vibration. As such, lipid rich structures of the skin were highlighted. From top (beginning of the movie) to bottom (end of the movie):

• Polygonal intercellular space of the stratum corneum
• Viable epidermis with hair follicle
• Sebaceous gland

The image stack was taken on fresh tissue, without any preparation or labeling.