Excitation parameters optimized for coherent anti-Stokes Raman scattering imaging of myelinated tissue

Abstract

Coherent anti-Stokes Raman scattering (CARS) generates a strong label-free signal in the long wavenumber C─H stretching region. Lipid-rich myelinated tissues, such as brain and spinal cord, would appear to be ideal subjects for imaging with CARS laser-scanning microscopy. However, the highly ordered, biochemically complex, and highly scattering nature of such tissues complicate the use of the technique. A CARS microscopy approach is presented that overcomes the challenges of imaging myelinated tissue to achieve chemically and orientationally sensitive high-resolution images.

Keywords: Raman scattering; coherent anti-Stokes Raman scattering; dispersion; myelin; nonlinear optics; polarization.

Fig. 1

Representative plots of phase and frequency for two broadband sources with (a) unmatched chirp (${\mathrm{IFD}}_{t0}\ne {\mathrm{IFD}}_{t1}$) and (b) matched chirp (${\mathrm{IFD}}_{t0}={\mathrm{IFD}}_{t1}$). Shaded area highlights frequency difference between the two sources at any instant.

Fig. 2

(a) Schematic representation of a lipid molecule illustrating orientation of hydrogen bonds relative to the carbon backbone of the hydrophobic tail. Large circle represents the hydrophilic polar head group. (b) Illustration of an ideal membrane bilayer highlighting the orientation of $\mathrm{C}─\mathrm{H}$ bonds parallel to the membrane surface.

Fig. 3

Schematic diagram of complete imaging system. From right: Dual NOPA, dual noncollinear optical parametric amplifier; Yb, ytterbium-doped fiber laser (detailed in Sec. 3.1); CB, chirping blocks for dispersion control (Sec. 3.2); ½ and ¼ $\lambda$, half- and quarter-waveplates for polarization control (Sec. 3.3); BS, beam splitter; BD, beam dump; BE, beam expander telescope composed of 30- and 100-mm focal-length achromatic lenses; DL, delay line; DCA, dichroic combiner “A” for combining pump and Stokes; DCB, dichroic combiner “B” for permitting optional introduction of the residual beam from the fiber laser; GSM, galvanometer scanning mirror pair; SL, scan lens; TL, tube lens; PDF, primary dichroic filter for separating NIR pump and Stokes beams from visible fluorescence and anti-Stokes signals; SDF, secondary dichroic filter for separating CARS from fluorescence or second harmonic signals; BPF, bandpass filters in interchangeable holders to select desired emission wavelength; and PC, personal computer for acquisition. (Filters and scanning mechanics are detailed in Sec. 3.4.)

Fig. 4

(a) Constant IFD between ${\omega }_{\mathrm{p}}$ and ${\omega }_{\mathrm{s}}$ at ${\tau }_0$ and (b) increasing IFD (${\mathrm{IFD}}^{\prime }$) by delaying arrival of ${\omega }_{\mathrm{s}}$ by time delay $\mathrm{\Delta }\tau$.

Fig. 5

(a) Published Raman spectra for PS, PE, and PMMA polymers. (b) Unprocessed CARS spectra acquired from polymer beads mounted in Sylgard 184. Delay axis refers to relative delay in fs between arrival of ${\omega }_{\mathrm{p}}$ and ${\omega }_{\mathrm{s}}$. Arrow at $-700\mathrm{fs}$ highlights contamination in the acquired spectra from Sylgard mounting media. (c) Unprocessed CARS spectra of Sylgard showing strong peak at $-700\mathrm{fs}$.

Fig. 6

False-color CARS image of dorsal column from wild-type mouse fixed with 4% PFA. Arrows denote features of interest including probable (a and b) Schmidt–Lanterman incisures and a (c) node of Ranvier. Submicron features are visible in the axon interiors including (d) small membrane folds and vesicular structures. This image was acquired with excitation polarization aligned with the direction of the axons which maximized signals from normal compacted myelin.

Fig. 7

DREZ of wild-type mouse spinal column (dorsal) imaged with polarized CARS excitation. Double-headed arrows indicate direction of linearly polarized ${\omega }_{\mathrm{p}}$ and ${\omega }_{\mathrm{s}}$ for each image.

Fig. 8

CARS spectra of CNS myelin for excitation polarization aligned (solid) and orthogonal (dashed) to the direction of the membrane (longitudinal axons). The three peaks are the ${\mathrm{CH}}_2$ symmetric (I), ${\mathrm{CH}}_2$ asymmetric (II), and ${\mathrm{CH}}_3$ vibrations (III).