Schematic of a laser-scanning CARS microscope that allows imaging with forward-detected CARS (F-CARS), epi-detected CARS (E-CARS), two-photon excitation fluorescence (TPEF), and sum frequency generation (SFG) signals produced by two synchronized pulsed lasers at frequencies ω_p and ω_s. The energy diagram of CARS is shown above the setup.

(A) Diagram shows the myelin sheath in the central nervous system. The myelin is a multiple-layer membrane formed by oligodendrocytes, containing a high lipid to protein ratio. MBP: myelin basic protein. PLP: proteolipid protein. (B) E-CARS spectrum of a single axonal myelin sheath measured by manually tuning the Stokes laser wavelength. The pump laser frequency was fixed at 14,081 cm⁻¹. (C) E-CARS image of myelin sheath wrapping around the axons when the Raman shift was tuned to 2840 cm⁻¹. Bar = 10 μm. (D) Reconstruction of 3D CARS imaging of myelin sheath in spinal tissue. The inset is XZ images showing the transverse section of myelin sheath. Bar = 10 μm. (E) In vivo E-CARS imaging of myelinated axons and SHG imaging of collagen fibrils surrounding sciatic nerve and between the axons. Bar = 20 μm.

Characterization of lyso-PtdCho-induced myelin swelling by CARS microscopy. For all images, bar = 10 μm. (A) CARS image of normal myelin sheath wrapping two parallel axons. (B) CARS image of partially swollen myelin sheath acquired at 5 min after injecting 2 μL of 10 mg/mL lyso-PtdCho into the tissue. (C) CARS intensity profiles of normal and swollen myelin fibers. Green: taken along the green line in (A). Red: taken along the red line in (B). The intensity profiles clearly showed that the CARS intensity from the swollen region was approximately 1/3 of that from the compact region, indicating a reduction of the lipid packing density in the membrane. (D and E) CARS images of normal myelin sheath and totally swollen myelin sheath with vertical (↕) and horizontal (↔) excitation polarization. (F) The ordering degree characterized by I_||/I_⊥ of intra- myelin lipids for normal and swollen myelin sheath. Here I_|| and I_⊥ are defined as the CARS intensities with the excitation polarization parallel with or perpendicular to the myelin length. The ratio of I_|| to I_⊥ measures the ordering degree of the myelin lipids. For healthy myelin along y-oriented axons (D), the average CARS intensity from the equatorial plane generated with y-polarized beams (I_||) is 3.53 (±0.09) times that with x-polarized beams (I_⊥). I_|| is larger than I_⊥ because the symmetry axis of the CH₂ groups in the equatorial plane of the myelin is perpendicular to the x direction. For the swollen myelin (E), the value of I_||/I_⊥ was measured to be 0.98 ± 0.09. Such independence on the excitation polarization (I_|| = I_⊥) indicates a complete degradation of the lamellar structure in the swollen myelin sheath. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

CARS images and spectra of intracellular lipid bodies. (A) Collocalized image of CARS (red) and TPEF (green) in a Y-1 Cell. CARS contrast comes from lipid droplets and TPEF contrast shows the mitochondria labeled by MitoTracker. (B) Trajectories of LDs over a period of 24 s. LDs underwent active transport movement of ~2 μm within 24 s. (C) Collocalized image of lipid droplets and mitochondria in 3T3-L1 cells shows very different distribution from Y-1 cells. Bars are 2 μm in (A)–(C) (from Ref. [23]. Used with permission). (D) CARS Spectra of wild-type and mutant C. elegans in vivo. (N2 L1 and L4: wild-type early and late stage larva; pha-3: feeding-deficient mutant nematode; Dauer: daf-4 mutant of promoted lipid store). Spectra at symmetric CH₂ vibration (2845 cm⁻¹) and asymmetric CH₂ vibration (2880 cm ⁻¹) indicate lipid order shifted from gel to liquid when lipid storage is promoted. (E) Resonant at 2845 cm⁻¹ and (F) nonresonant at 2790 cm⁻¹ images of lipid storage in wild-type late-stage (N2 L4) larva in vivo. Bars are 10 μm in (E) and (F) (from Ref. [35]. Used with permission). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Cross-sectional CARS (gray), SFG (blue), and TPEF (green) images of a coronary artery bearing early atherosclerotic lesion. (A) CARS image of the arterial wall. The red square shows thickened intima with scattered lipid rich cells. (B) Collagen distribution of the arterial wall imaged by SFG. (C) Elastic lamellae imaged by TPEF. Bar = 300 μm for (A)–(C). (D) Zoomed-in image at the red square in (A) shows the distribution of lipid-rich cells within collagen matrix in thickened intima. Bar = 30 μm in (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)