Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues

Abstract

Sum frequency generation (SFG) and second harmonic generation (SHG) were observed from helical fibrils in spinal cord white matter isolated from guinea pigs. By combining SFG with coherent anti-Stokes Raman scattering microscopy, which allows visualization of myelinated axons, these fibers were found to be distributed near the surface of the spinal cord, between adjacent axons, and along the blood vessels. Using 20-microm-thick tissue slices, the ratio of forward to backward SHG signal from large bundles was found to be much larger than that from small single fibrils, indicating a phase-matching effect in coherent microscopy. Based on the intensity profiles across fibrils and the size dependence of forward and backward signal from the same fibril, we concluded that the main SHG signal directly originates from the fibrils, but not from surface SHG effects. Further polarization analysis of the SHG signal showed that the symmetry property of the fibril could be well described with a cylindrical model. Colocalization of the SHG signal with two-photon excitation fluorescence (TPEF) from the immunostaining of glial fibrillary acidic protein demonstrated that SHG arises from astroglial filaments. This assignment was further supported by colocalization of the SHG contrast with TPEF signals from astrocyte processes labeled by a Ca(2+) indicator and sulforhodamine 101. This work shows that a combination of three nonlinear optical imaging techniques--coherent anti-Stokes Raman scattering, TPEF, and SHG (SFG) microscopy--allows simultaneous visualization of different structures in a complex biological system.

FIGURE 1

Schematic of a multimodal nonlinear optical imaging system. Two colinearly combined 2.5-ps Ti:Sapphire laser beams were used for CARS and SFG imaging. A 200-fs Ti:Sapphire laser was used for SHG and TPEF imaging. A flip mirror was used to switch between the femtosecond and picosecond laser sources. Images were acquired by scanning a pair of galvanometer mirrors. A water immersion objective (NA 1.2) was used to focus the laser beams into a sample. The NLO signals were detected in either a forward or a backward direction. An air condenser (NA 0.55) was used to collect the forward CARS signal from ex vivo spinal tissues. A water immersion objective (NA 1.2) was used to collect the forward SHG signal from thin tissue slices. (D) dichroic mirror; (F) flip mirror.

FIGURE 2

SFG and SHG imaging of helical fibers (green) and CARS imaging of myelin sheath (red) in ex vivo spinal tissues. (A) SFG image near the surface of spinal cord white matter. Scale bar, 10 μm. (B) Overlaid SFG image of fibrils and CARS image of myelinated axons inside spinal cord white matter. (Inset) Overlay of the SFG and CARS images of a node of Ranvier surrounded by astrocyte processes. Scale bar, 5 μm. (C) CARS (red) and SHG (green) intensity profiles along the line indicated by the arrow in B. (D and E) CARS (red) and SFG (green) images of a perivascular area containing a branched blood vessel. (D, inset) Overlaid CARS and SFG images. Scale bar, 20 μm. (E) The blood vessel is indicated by dashed white lines. (F) SHG image of fibrils in the same location of spinal cord white matter as in B. Scale bar, 10 μm. The average laser power for SFG and CARS imaging was around 10 mW at the sample. The average laser power for SHG imaging was 11.2 mW at the sample.

FIGURE 3

Characterization of the SFG and SHG signals from ex vivo spinal tissues. (A) Linear dependence of SFG intensity on pump laser power with fixed Stokes power at 3.5 mW at the sample. The squares represent the data and the solid line represents linear fit. (B) Linear dependence of SFG intensity on Stokes laser power with fixed pump power at 8.5 mW at the sample. The solid circles represent the data and the solid line represents linear fit. (C) Emission spectra of SHG and SFG produced by two picosecond beams at 705.8 nm (average power 3.96 mW at the sample) and 882.6 nm (average power 1.12 mW at the sample), respectively. (D) SHG excitation profile generated by tuning the 200-fs laser in the region of 700 to 900 nm. The SHG intensity was normalized by the transmission of the microscope components (dichroic mirrors, objective, and filters) and the laser power. The error bar for each point represents the standard error of three measurements.

FIGURE 4

Simultaneously acquired forward and backward SHG images in a 20-μm-thick spinal tissue slice. The forward signal was collected by a 60× water immersion objective. The forward and backward PMT detectors were of the same type and set at the same gain. (A and B) Simultaneously acquired forward and backward SHG images of fibrils inside spinal cord white matter. (C) Line analysis of a large fibril indicated by red lines in A and B. (D and E) Simultaneously acquired forward and backward SHG images of the same area as in A and B but at a different depth. (F) Line analysis of a small fibril indicated by red lines in D and E. The laser power at the sample was 7 mW. Scale bar, 10 μm.

FIGURE 5

Polarization analysis of forward SHG signal. The scheme of each polarization measurement is shown in the left panel and the result is shown in the right panel. The fiber axis is defined as the y axis and the beam propagation is along the z axis. θ, angle between the excitation field and the y axis; ϕ, angle between the emission polarizer axis and the y axis; E₁, excitation field; P, axis of the emission polarizer before the detector. (A–C) Data points are shown as squares with error bars, and solid curves are the theoretical fit. (A) Excitation polarization dependence of the y-polarized SHG component. The least-squares fitting with Eq. 2a gives where ρ₁ =2.13. (B) Excitation polarization dependence of the x-polarized SHG component. The fitting with Eq. 2b gives (C) Excitation polarization dependence of total SHG intensity. The fitting with Eq. 1 gives where ρ₁ =2.13 and ρ₂ =1.62. (D) The detected intensity of the y-polarized excitation laser beam (solid curve) and SHG emission (squares with error bars) with respect to ϕ. SHG emission data points indicate that it was also linearly polarized along the y axis. For all measurements, the laser power at the sample was 7 mW. For each data point, SHG intensities from a single fibril were normalized by its peak intensity, and the normalized intensities from different fibrils were used to calculate the mean value and standard deviation. Because the peak intensities of I_x, I_y, and I_total are at different angles, the amplitude coefficients (0.23, 1.03, and 0.22, respectively) after normalization do not represent I_p, and I_p in Eqs. 2a, 2b, and 1, respectively.

FIGURE 6

Colocalization of SHG from the fibrils and TPEF from immunolabeled GFAP. (A and B) SHG and TPEF images, respectively, of a fixed spinal tissue labeled with Cy3-anti-GFAP serum. (C and D) SHG and TPEF images, respectively, of a control sample without antiserum Cy3. The femtosecond laser power used for TPEF and SHG was 14 mW at the sample. Bar = 10 μm.

FIGURE 7

Colocalization of SFG or SHG signal from fibrils (green) and TPEF signal from Ca²⁺ indicator (blue) or SR101-labeled astrocyte processes (gray) in ex vivo spinal tissues. (A) TPEF image of OG (a calcium indicator). (B) SFG image of the same location as in A. The red in A and B represents the CARS contrast from myelin sheath. (C) TPEF image of the surface of a spinal tissue sample. (D) Simultaneously acquired SFG image of the same location as in C. (E) TPEF image of SR101-labeled spinal tissue. (F) Simultaneously acquired SHG image of the same location as in E. The power used for TPEF and SHG was 8.4 mW at the sample. (A–D) Scale bar, 10 μm. (E and F) Scale bar, 5 μm.