A new publication from Opto-Electronic Advances; DOI 10.29026/oea.2024.240064, discusses advanced phase-controlled 3D biochemical imaging.
CARS microscopy was the first to be developed but faced challenges like distorted Raman spectra and sensitivity issue due to non-resonant background interference. SRS microscopy overcame these hurdles, enabling highly sensitive, quantitative biochemical imaging by avoiding non-resonant background interference. In recent years, SRS has been widely used in various fields, such as cancer diagnosis and characterization, tumor metabolisms, drug deliveries and pharmacodynamics, molecular genetics, organ functions, and developmental biology. In SRS, the two laser beams (pump and Stokes) are spatially and temporally combined and focused onto a sample. When their frequency difference matches the target molecule's vibration, coherent Raman scattering process will occur along the phase-matching direction, empowering SRS imaging with biomolecular contrast.
The authors of this article have developed an advanced SRS 3D microscopy called phase-controlled SRS (PC-SRS). This new technique allows for rapid and deep tissue 3D chemical imaging without the need for mechanical z-scanning. PC-SRS uses unique imaging system designs with the combination of ring-shaped pump beam and Gaussian Stokes beam (Fig. 1a), and the incorporation of Zernike polynomials (ZPs) in Fig. 1(b-d). These designs allow for the precise engineering of the Bessel beam's length and the corrections of the imaging system aberrations in both beams. PC-SRS possess a high signal-to-noise ratio (SNR) after compressing the length of Bessel beam and correcting the distortions for each beam. By electronically tuning phase patterns on the spatial light modulator (SLM), they can control the beam positions axially within the tissue, enabling 3D imaging even in thick samples (Fig. 1e-g).
PC-SRS has shown significant improvements in imaging speed and depth. For example, it can monitor the Brownian motions of polymer beads in water at high speeds (77 ms intervals, 13 Hz volume rate), and it offers deeper imaging capabilities in highly scattering media like brain tissue. Using scattering-resilient Bessel pump beam and longer wavelength Stokes beam (1041 nm in the NIR-II window), PC-SRS achieved about double the imaging depth compared to conventional SRS technique. Additionally, the authors used PC-SRS to study metabolic activities of liver tumor in living zebrafish. By tracking the formation of C–D bonds in macromolecules synthesized during cellular metabolic activities, they observed higher metabolic activity in tumor tissues compared to normal ones. The rapid depth-resolved imaging capabilities of PC-SRS allowed them to unravel how metabolic activity varied at different tissue depths, i.e., a highly active metabolism being observed in shallower liver regions compared to the deeper areas in liver tumor.
The authors believe that PC-SRS holds great promise for real-time monitoring of live cells and tissues, facilitating the transformation of the understandings of their metabolic and dynamic processes in live cells and tissue into the fields like cancer research, drug delivery, and developmental biology.
Keywords: SRS 3D imaging / phase-controlled light focusing / image aberration corrections / deep tissue imaging
