Research ArticleOPTICAL MICROSCOPY

Single-frame 3D fluorescence microscopy with ultraminiature lensless FlatScope

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Science Advances  08 Dec 2017:
Vol. 3, no. 12, e1701548
DOI: 10.1126/sciadv.1701548
  • Fig. 1 Traditional microscope versus FlatScope.

    (A) Traditional microscopes capture the scene through an objective and tube lens (~20 to 460 mm), resulting in a quality image directly on the imaging sensor. (B) FlatScope captures the scene through an amplitude mask and spacer (~0.2 mm) and computationally reconstructs the image. Scale bars, 100 μm (inset, 50 μm). (C) Comparison of form factor and resolution for traditional lensed research microscopes, GRIN lens microscope, and FlatScope. FlatScope achieves high-resolution imaging while maintaining a large ratio of FOV relative to the cross-sectional area of the device (see Materials and Methods for elaboration). Microscope objectives are Olympus MPlanFL N (1.25×/2.5×/5×, NA = 0.04/0.08/0.15), Nikon Apochromat (1×/2×/4×, NA = 0.04/0.1/0.2), and Zeiss Fluar (2.5×/5×, NA = 0.12/0.25). (D) FlatScope prototype (shown without absorptive filter). Scale bars, 100 μm.

  • Fig. 2 T2S model.

    (A) Illustration of the FlatScope model using a single fluorescent bead as the scene. (B) Fluorescent point source at depth d, represented as input image Xd. (C) FlatScope measurement Y. FlatScope measurement can be decomposed as a superposition of two patterns: (D) pattern when there is no mask in place (open) and (E) pattern due to the coding of the mask. Each of the patterns is separable along x and y directions and can be written as (F and G) two separable transfer functions. The FlatScope model, which we call as the T2S, is the superposition of the two separable transfer functions.

  • Fig. 3 Resolution tests with the FlatScope prototype.

    (A) Double slit with a 1.6-μm gap imaged with a 10× objective. (B) Captured FlatScope image. (C) FlatScope reconstruction of the double slit with a 1.6-μm gap. (D to F) FlatScope reconstructions of USAF resolution target at distances from the mask surface of 200 μm (D), 525 μm (E), and 1025 μm (F). Scale bar, 100 μm.

  • Fig. 4 3D volume reconstruction of 10-μm fluorescent beads suspended in agarose.

    (A) FlatScope reconstruction as a maximum intensity projection along the z axis as well as a ZY slice (blue box) and an XZ slice (red box). (B) Estimated 3D positions of beads from the FlatScope reconstruction. (C) Ground truth data captured by confocal microscope (10× objective). (D) Depth profile of reconstructed beads compared to ground truth confocal images. Empirically, we can see that the axial spread of 10-μm beads is around 15 μm in FlatScope reconstruction. That is, FlatScope’s depth resolution is less than 15 μm. The three beads shown are at depths of 255, 270, and 310 μm from the top surface (filter) of the FlatScope.

  • Fig. 5 3D volumetric video reconstruction of moving 10-μm fluorescent beads.

    (A) Subsection of FlatScope time-lapse reconstruction of 3D volume with 10-μm beads flowing in microfluidic channels (approximate location of channels drawn to highlight bead path and depth). Scale bar, 50 μm (FlatScope prototype graphic at the top not to scale). (B) Captured images of frames 1, 3, and 5 (false-colored to match time progression). (C and D) FlatScope reconstructions of frames 1, 3, and 5 at estimated depths of 265 and 355 μm, respectively (dashed lines indicate approximate location of microfluidic channels). Reconstructed beads false-colored to match time progression. Scale bar, 50 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/12/e1701548/DC1

    section S1. Derivation of T2S model

    section S2. Computational tractability

    section S3. Model calibration

    section S4. Gradient direction for iterative optimization

    section S5. MURA: A separable pattern

    section S6. FlatCam model: An approximation to the T2S model

    section S7. Diffraction effects and T2S model

    section S8. Light collection

    section S9. Impact of sensor saturation

    fig. S1. Calibration setup.

    fig. S2. Digital focusing.

    fig. S3. Simulation comparison of bare sensor, FlatScope, and FlatCam.

    fig. S4. 3D volume reconstruction accuracy.

    fig. S5. Fabrication of FlatScope.

    fig. S6. Refractive index matching.

    fig. S7. T2S derivation.

    fig. S8. Aberration removal using RPCA.

    fig. S9. Removing effects from autofluorescence.

    fig. S10. T2S model error from diffraction.

    fig. S11. Light collection comparison of FlatScope and microscope objectives.

    movie S1. Digital focusing of simulated resolution target.

    movie S2. Fluorescent beads flowing in microfluidic channels of different depths.

  • Supplementary Materials

    This PDF file includes:

    • section S1. Derivation of T2S model
    • section S2. Computational tractability
    • section S3. Model calibration
    • section S4. Gradient direction for iterative optimization
    • section S5. MURA: A separable pattern
    • section S6. FlatCam model: An approximation to the T2S model
    • section S7. Diffraction effects and T2S model
    • section S8. Light collection
    • section S9. Impact of sensor saturation
    • fig. S1. Calibration setup.
    • fig. S2. Digital focusing.
    • fig. S3. Simulation comparison of bare sensor, FlatScope, and FlatCam.
    • fig. S4. 3D volume reconstruction accuracy.
    • fig. S5. Fabrication of FlatScope.
    • fig. S6. Refractive index matching.
    • fig. S7. T2S derivation.
    • fig. S8. Aberration removal using RPCA.
    • fig. S9. Removing effects from autofluorescence.
    • fig. S10. T2S model error from diffraction.
    • fig. S11. Light collection comparison of FlatScope and microscope objectives.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.mp4 format). Digital focusing of simulated resolution target.
    • movie S2 (.mp4 format). Fluorescent beads flowing in microfluidic channels of different depths.

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