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Wide-field multiphoton imaging through scattering media without correction

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Science Advances  12 Oct 2018:
Vol. 4, no. 10, eaau1338
DOI: 10.1126/sciadv.aau1338
  • Fig. 1 Working principle of TRAFIX.

    (A) A femtosecond laser beam is expanded onto a spatial light modulator (SLM) that generates Hadamard patterns. Subsequently, the beam is diffracted from a grating, and the Hadamard patterns are projected onto a fluorescent sample after propagating through a scattering medium. Fluorescent light emitted by the sample is collected by the same objective after passing through the scattering medium a second time (epifluorescence geometry), and the total intensity is measured by a single-pixel detector. (B) A TF beam propagates through a turbid medium with minimal distortion, retaining the integrity of illumination patterns in the sample plane. Emitted fluorescent photons scatter as they propagate back through the tissue. In contrast to standard TF microscopy, TRAFIX tolerates scrambling of back-propagating light since only an intensity measurement is performed. In a single-pixel measurement, the fluorescent target is sequentially illuminated with Hadamard patterns (ψn), and the total intensity detected is stored as a coefficient (ωn). Gray background in the second column denotes regions of zero intensity. By adding up the Hadamard patterns weighted by their respective coefficients, an image of the fluorescent sample is reconstructed.

  • Fig. 2 Images of fluorescent microscopic samples through scattering phantoms.

    Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 500- and 540-μm of scattering phantoms, respectively. (A and F) Images taken from the reference imaging system under uniform TF illumination across the FOV. Exposure time was set to 20 s, and camera binning was 4 × 4 (beads) and 2 × 2 (cells). (B to E, G, and H) Images obtained in epifluorescence configuration with TRAFIX using a Hadamard basis containing 4096 illumination patterns. They were reconstructed with different CRs corresponding to 100% (CR = 1), 50% (CR = 2), 25% (CR = 4), or 12.5% (CR = 8) of the total patterns. Each measurement under individual illumination patterns was taken with a binning of 64 × 64 and an exposure time of 0.5 s. The spacing between beads was measured in all five images obtaining deviations smaller than 3% from the reference image (table S3). The diameters of the cells in (F) were measured to be 20.7 and 14.3 μm, respectively, and their values in (G) and (H) differ less than 4 and 12% from the reference value (table S2). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

  • Fig. 3 Images of fluorescent microscopic samples through unfixed human colon tissue.

    Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 250 and 200 μm of human colon tissue, respectively. (A and C) Images taken from the reference imaging system under uniform TF illumination across the FOV. Camera binning in (A) was set to 4 × 4, and exposure time was 5 s. No camera binning was used in (C), and exposure time was 15 s. (B and D) Images obtained with TRAFIX using a Hadamard basis containing 1024 and 4096 illumination patterns, respectively. All patterns were used for image reconstruction (CR = 1). Camera binning for each Hadamard pattern was set to 64 × 64, and exposure time values were (B) 1 s and (D) 0.75 s. The spacing between beads and the diameter of cells were measured to assess image quality (tables S3 and S2, respectively). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

  • Fig. 4 Comparison of a hidden object and the retrieved images through fixed rat brain tissue.

    (A) Reference image of a fluorescent micropattern without any scattering sample. (B) Image obtained by conventional TF microscopy (i.e., under uniform wide-field TF illumination with wide-field detection in epifluorescence configuration) through 400 μm of fixed rat brain tissue. (C and D) Reconstructed images obtained with TRAFIX through 200 and 400 μm of rat brain tissue, respectively. The two retrieved images were reconstructed using a full Hadamard basis containing 1024 patterns. Camera binning was set to 64 × 64, and exposure time values were (C) 0.2 s and (D) 1 s. Small intensity variations in the reconstructed images arise from inhomogeneities in the fluorescent micropattern originated in the imprinting process. Larger intensity variations are due to inhomogeneities in light transmission through the highly anisotropic scattering medium. This also applies to figs. S7, S9, S10, and S12. The SBR is shown for all reconstructed images. Scale bar, 10 μm.

  • Fig. 5 Numerical simulation of TRAFIX in scattering media.

    (A) Simulated TF laser beams at the focal plane through a 400-μm-thick brain tissue. The solid red curve indicates the smoothed-out lateral beam profile, taking all monochromatic components of the laser pulse into account. (B) Total fluorescence power collected with an NA = 0.8 microscope objective for different thicknesses of brain tissue. Incident laser power at sample surface is set to 100 arbitrary units (a.u.).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/10/eaau1338/DC1

    Note S1. Numerical simulations

    Note S2. Single-pixel detection and compressive sensing

    Note S3. Microscope characterization

    Note S4. Comparison between TRAFIX and point-scanning two-photon microscopy (2PM)

    Note S5. Polarization state evaluation

    Fig. S1. Numerically simulated TF laser beam propagating through 400 μm of brain tissue.

    Fig. S2. Properties of a numerically simulated TF laser beam through brain tissue.

    Fig. S3. Effect of scattering on the beam profile with and without TF.

    Fig. S4. Depth profile of a TF beam through a scattering phantom.

    Fig. S5. Characterization of a TF beam through a scattering phantom.

    Fig. S6. Images of fluorescent microscopic samples without scattering.

    Fig. S7. Comparison of a hidden object and retrieved images through a scattering phantom with different resolution.

    Fig. S8. Image of 4.8-μm fluorescent beads in a volumetric scattering phantom.

    Fig. S9. Comparison of SBR of TRAFIX and point-scanning two-photon microscopy (2PM) at depth.

    Fig. S10. Comparison of TRAFIX and point-scanning two-photon microscopy (2PM) through human colon tissue.

    Fig. S11. Axial confinement in TRAFIX and point-scanning two-photon microscopy (2PM).

    Fig. S12. Photobleaching comparison of TRAFIX and point-scanning two-photon microscopy (2PM).

    Fig. S13. Effect of scattering on illumination beams in point-scanning two-photon microscopy (2PM) and TRAFIX.

    Fig. S14. Effect of turbid media on light polarization.

    Table S1. SBR measured for all the images shown in this work.

    Table S2. Cell diameters of all images shown in this work.

    Table S3. Beads spacing corresponding to all images shown in this work.

    References (5561)

  • Supplementary Materials

    This PDF file includes:

    • Note S1. Numerical simulations
    • Note S2. Single-pixel detection and compressive sensing
    • Note S3. Microscope characterization
    • Note S4. Comparison between TRAFIX and point-scanning two-photon microscopy (2PM)
    • Note S5. Polarization state evaluation
    • Fig. S1. Numerically simulated TF laser beam propagating through 400 μm of brain tissue.
    • Fig. S2. Properties of a numerically simulated TF laser beam through brain tissue.
    • Fig. S3. Effect of scattering on the beam profile with and without TF.
    • Fig. S4. Depth profile of a TF beam through a scattering phantom.
    • Fig. S5. Characterization of a TF beam through a scattering phantom.
    • Fig. S6. Images of fluorescent microscopic samples without scattering.
    • Fig. S7. Comparison of a hidden object and retrieved images through a scattering phantom with different resolution.
    • Fig. S8. Image of 4.8-μm fluorescent beads in a volumetric scattering phantom.
    • Fig. S9. Comparison of SBR of TRAFIX and point-scanning two-photon microscopy (2PM) at depth.
    • Fig. S10. Comparison of TRAFIX and point-scanning two-photon microscopy (2PM) through human colon tissue.
    • Fig. S11. Axial confinement in TRAFIX and point-scanning two-photon microscopy (2PM).
    • Fig. S12. Photobleaching comparison of TRAFIX and point-scanning two-photon microscopy (2PM).
    • Fig. S13. Effect of scattering on illumination beams in point-scanning two-photon microscopy (2PM) and TRAFIX.
    • Fig. S14. Effect of turbid media on light polarization.
    • Table S1. SBR measured for all the images shown in this work.
    • Table S2. Cell diameters of all images shown in this work.
    • Table S3. Beads spacing corresponding to all images shown in this work.
    • References (5561)

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