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Wang WQ, Huang ZW. Stimulated Raman scattering microscopy with phase-controlled light focusing and aberration correction for rapid and label-free, volumetric deep tissue imaging. Opto-Electron Adv 7, 240064 (2024). doi: 10.29026/oea.2024.240064
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Stimulated Raman scattering microscopy with phase-controlled light focusing and aberration correction for rapid and label-free, volumetric deep tissue imaging
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Abstract
We report a novel stimulated Raman scattering (SRS) microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid, deep tissue 3D chemical imaging with subcellular resolution. To accomplish phase-controlled SRS (PC-SRS), we utilize a single spatial light modulator to electronically tune the axial positioning of both the shortened-length Bessel pump and the focused Gaussian Stokes beams, enabling z-scanning-free optical sectioning in the sample. By incorporating Zernike polynomials into the phase patterns, we simultaneously correct the system aberrations at two separate wavelengths (~240 nm difference), achieving a ~3-fold enhancement in signal-to-noise ratio over the uncorrected imaging system. PC-SRS provides >2-fold improvement in imaging depth in various samples (e.g., polystyrene bead phantoms, porcine brain tissue) as well as achieves SRS 3D imaging speed of ~13 Hz per volume for real-time monitoring of Brownian motion of polymer beads in water, superior to conventional point-scanning SRS 3D imaging. We further utilize PC-SRS to observe the metabolic activities of the entire tumor liver in living zebrafish in cell-silent region, unraveling the upregulated metabolism in liver tumor compared to normal liver. This work shows that PC-SRS provides unprecedented insights into morpho-chemistry, metabolic and dynamic functioning of live cells and tissue in real-time at the subcellular level. -
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References
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Wang WQ, Huang ZW. Stimulated Raman scattering microscopy with phase-controlled light focusing and aberration correction for rapid and label-free, volumetric deep tissue imaging. Opto-Electron Adv 7, 240064 (2024). doi: 10.29026/oea.2024.240064
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Figure 1.
(a) Schematic of the PC-SRS microscope system for 3D chemical imaging. EOM, electro-optical modulator; DM, dichroic mirror; SLM, spatial light modulator; Ape, aperture; GM, galvo mirror; SL, scan lens; TL, tube lens; F, filter; PD, photodiode; LIA, lock-in amplifier. Inset drawings illustrate the ring-shaped pump beam and Gaussian Stokes beam on the SLM. (b) The distortion-corrected phase pattern for the SLM. (c, d) The phase patterns mimicking the ZPs for pump and Stokes beams, respectively, which are incorporated into (b) to correct system aberrations. (e) The phase patterns used in experiments to control the axial positionings of the Bessel pump and Gaussian Stokes beams in the sample. The region enclosed by the solid red circle is designated for the Stokes beam, and the area between the solid red and yellow dashed circles is for the pump beam. (f) The schematic of the generated shortened-length Bessel pump beam and Gaussian Stokes beam at different depths corresponding to the phase patterns in (e). (g) Raw SRS images of 10 μm PS beads (2885 cm−1 of CH2 asymmetric stretching) acquired by PC-SRS at z=0 μm, 8 μm, and 16 μm, respectively. Scale bar: 20 μm.
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Figure 2.
(a) SRS image (2885 cm−1 for CH2 asymmetric stretching) of 1 μm PMMA beads using PC-SRS with AC-ON. Scale bar: 2 μm. (b) SRS image of the same PMMA beads using PC-SRS with AC-OFF. Scale bar: 2 μm. (c) SRS intensities of PMMA beads (indicated by yellow arrows) along the x-direction in (a) and (b). (d) The relationship between the SRS intensities and DMSO concentrations, where the detection limit of PC-SRS (39 mM, at SNR=1) is calculated with a linear fit curve. (e) SRS image (2885 cm−1) of a 500 nm PS bead in the x-y plane using PC-SRS. Scale bar: 250 nm. (f) SRS image of the 500 nm PS bead in the x-z plane. Scale bar: 2 μm. The average powers of the pump and Stokes beams on the beads are 20 mW and 45 mW, respectively. (g–h) SRS intensity distributions along the x- and z-directions in (e) and (f), with the estimated FWHMs (lateral: 0.52 μm and axial: 5.7 μm) for PC-SRS.
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Figure 3.
(a, b) SRS 3D images of 10 μm PS beads in a gel phantom (2885 cm−1, CH2 asymmetric stretching) using PC-SRS and C-SRS imaging. Yellow arrows highlight the brighter PS beads in deeper regions captured by PC-SRS. Image volume: 72 μm × 72 μm × 120 μm. Axial step size of 8.5 μm. A total of 14 depths are imaged with a 2.3 s acquisition time per 3D volume. Scale bar: 20 μm. (c) Comparison of normalized SRS intensities of PS beads at various depths by PC-SRS and C-SRS. SRS intensities at different depths are normalized to the top layer (z=0) for better comparison. (d, e) SRS 3D images (2885 cm−1) of porcine brain tissue acquired by PC-SRS and C-SRS. Scale bar: 20 μm. Image volume: 184.6 μm × 184.6 μm × 80 μm, with an axial step size of 8.8 μm. A total of 9 depths are imaged with a 1.5 s acquisition time per 3D volume. Average pump beam powers are 20 mW for PC-SRS and 4 mW for C-SRS, with a Stokes beam power of 45 mW on the porcine brain. (f) Comparison of SRS intensities at each tissue depth in porcine brain between PC-SRS and C-SRS. SRS intensities at each depth are normalized to the top-layer (z=0 μm) for performance comparison. (g–i) Dynamic SRS 3D images (2885 cm−1) of 4.5 μm PS beads in water. Scale bar: 5 μm. Image volume: 23.08 μm × 23.08 μm × 16 μm, with an axial step size of 4 μm. A total of 4 depths are imaged with a 77 ms acquisition time per volume (13 Hz). Average powers of the pump and Stokes beams on the bead are 13 mW and 18 mW, respectively.
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Figure 4.
(a, b) SRS 3D images of tumor liver in zebrafish from 6 dpf to 11 dpf using PC-SRS (2935 cm−1, CH3 stretching of lipids and proteins, and 2186 cm−1, CD bond). Image volume: 184 μm × 184 μm × 80 μm. Scale bar: 50 μm. (c, d) SRS 3D images of normal liver in zebrafish from 6 dpf to 11 dpf (2935 cm−1 and 2186 cm−1), serving as normal control in comparison with (a, b). The regions surrounded by the red dotted lines represent the zebrafish liver acquired by PC-SRS. Image volume: 184 μm × 184 μm × 80 μm. Scalebar: 50 μm. The axial step size is 8.8 μm, with a total of 9 depths and a 15 s acquisition time for one 3D volume. Average laser powers were 20 mW at 797 nm and 25 mW at 848 nm, with a Stokes beam (1041 nm) power of 45 mW on the liver region. (e) Ratios of the volume-average SRS intensity at CD bond (2186 cm−1) to CH3 (2935 cm−1) over time in both tumor and normal liver in zebrafish. (f) Ratios of the CD bond to CH3 at various tissue depths in tumor and normal liver at 7 and 8 dpf.
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Figure 5.
(a, b) SRS 3D images of tumor liver in living zebrafish (7 dpf) captured at 2935 cm−1 (CH3 stretching of lipids and proteins), 2845 cm−1 (CH2 symmetric stretching of lipids), and 2186 cm−1 (CD bond) using PC-SRS. The protein information is obtained by subtracting the 3D images at 2845 cm−1 from those at 2935 cm−1. Image volume: 184 μm × 184 μm × 112 μm. Scale bar: 40 μm. (c, d) SRS 3D images of zebrafish normal liver (7 dpf) for the comparison with (a−b). Image volume: 184 μm × 184 μm × 112 μm. Scale bar: 40 μm. The axial step size is 8 μm, with a total of 14 depths and a 23 s acquisition time for one 3D volume. The average powers are 20 mW at 797 nm, 20 mW at 804 nm, and 25 mW at 848 nm, with a Stokes beam (1041 nm) power of 45 mW on the liver region. (e) Ratios of the volume-average CD bond intensity to CH intensity of lipids and proteins, as well as to lipids and proteins separately, in both tumor and normal liver. The ratio of CH3 intensity of proteins to CH2 intensity of lipids in both tumor and normal liver is also presented for comparison. (f) Comparison of the ratios of CD bond intensity to CH intensity versus the ratios of inherent CH3 protein intensity to CH2 lipid intensity between tumor and normal liver in zebrafish larvae.
