Comparing the fundamental imaging depth limit of two-photon, three-photon, and non-degenerate two-photon microscopy
Xiaojun Cheng, Sanaz Sadegh, Sharvari Zilpelwar, Anna Devor, Lei Tian, and David A. Boas
Vol. 45, Issue 10, pp. 2934-2937 (2020).
We have systematically characterized the degradation of imaging quality with depth in deep brain multi-photon microscopy, utilizing our recently developed numerical model that computes wave propagation in scattering media. The signal-to-background ratio (SBR) and the resolution determined by the width of the point spread function are obtained as functions of depth. We compare the imaging quality of two-photon (2PM), three-photon (3PM), and non-degenerate two-photon microscopy (ND-2PM) for mouse brain imaging. We show that the imaging depth of 2PM and ND-2PM are fundamentally limited by the SBR, while the SBR remains approximately invariant with imaging depth for 3PM. Instead, the imaging depth of 3PM is limited by the degradation of the resolution, if there is sufficient laser power to maintain the signal level at large depth. The roles of the concentration of dye molecules, the numerical aperture of the input light, the anisotropy factor , noise level, input laser power, and the effect of temporal broadening are also discussed.
Single-Shot 3D Widefield Fluorescence Imaging with a Computational Miniature Mesoscope
Yujia Xue, Ian G. Davison, David A. Boas, Lei Tian
Fluorescence imaging is indispensable to biology and neuroscience. The need for large-scale imaging in freely behaving animals has further driven the development in miniaturized microscopes (miniscopes). However, conventional microscopes / miniscopes are inherently constrained by their limited space-bandwidth-product, shallow depth-of-field, and the inability to resolve 3D distributed emitters. Here, we present a Computational Miniature Mesoscope (CM
Design of a high-resolution light field miniscope for volumetric imaging in scattering tissue
Yanqin Chen, Bo Xiong, Yujia Xue, Xin Jin, Joseph Greene, and Lei Tian
Biomedical Optics Express 11, pp. 1662-1678 (2020).
Integrating light field microscopy techniques with existing miniscope architectures has allowed for volumetric imaging of targeted brain regions in freely moving animals. However, the current design of light field miniscopes is limited by non-uniform resolution and long imaging path length. In an effort to overcome these limitations, this paper proposes an optimized Galilean-mode light field miniscope (Gali-MiniLFM), which achieves a more consistent resolution and a significantly shorter imaging path than its conventional counterparts. In addition, this paper provides a novel framework that incorporates the anticipated aberrations of the proposed Gali-MiniLFM into the point spread function (PSF) modeling. This more accurate PSF model can then be used in 3D reconstruction algorithms to further improve the resolution of the platform. Volumetric imaging in the brain necessitates the consideration of the effects of scattering. We conduct Monte Carlo simulations to demonstrate the robustness of the proposed Gali-MiniLFM for volumetric imaging in scattering tissue.
Development of a beam propagation method to simulate the point spread function degradation in scattering media
Xiaojun Cheng, Yunzhe Li, Jerome Mertz, Sava Sakadžić, Anna Devor, David A. Boas, Lei Tian
Opt. Lett. 44, 4989-4992 (2019).
Scattering is one of the main issues that limit the imaging depth in deep tissue optical imaging. To characterize the role of scattering, we have developed a forward model based on the beam propagation method and established the link between the macroscopic optical properties of the media and the statistical parameters of the phase masks applied to the wavefront. Using this model, we have analyzed the degradation of the point-spread function of the illumination beam in the transition regime from ballistic to diffusive light transport. Our method provides a wave-optic simulation toolkit to analyze the effects of scattering on image quality degradation in scanning microscopy. Our open-source implementation is available at https://github.com/BUNPC/Beam-Propagation-Method.