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Light-sheet microscopy with attenuation-compensated propagation-invariant beams

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Science Advances  06 Apr 2018:
Vol. 4, no. 4, eaar4817
DOI: 10.1126/sciadv.aar4817
  • Fig. 1 Principle of attenuation-compensation for an Airy light sheet.

    Ray optics representations of Airy light-sheet formation without (A) and with (B) attenuation (Cattn = 65 cm−1) and with attenuation-compensation (C; σ = 0.54). (D to F) Wave optical simulations of light-sheet profiles, (G to I) peak transverse intensity as a function of longitudinal coordinate, and (J to L) axial MTF thresholded at 5% contrast, respectively, for the light sheets shown in (A) to (C). Green and red lines in (L) match the 5% contour in (J) and (K), respectively. a.u., arbitrary units. (M to R) Simulated, recorded, and deconvolved images of the University crest and (S to U) simulated deconvolved images of a 1D resolution target, respectively, for the light sheets shown in (A) to (C). Pink solid lines in (P) to (R) indicate the edge of the FOV from theory. One-dimensional resolution target (P to R) has linewidth/spacing: 2 μm (top) and 1, 0.6, and 0.2 μm (bottom). (V and W) Intensity profiles through the dashed lines at x = 50 and 125 μm in (S) to (U). Simulation parameters were set to mirror experimental parameters (see Materials and Methods).

  • Fig. 2 Principle of attenuation-compensation for a Bessel light sheet.

    Simulated xz intensity profiles for a flat-top Bessel beam without (A) and with (B) attenuation (Cattn = 65 cm−1) and with partial (C; σB = 0.11) and full (D; σB = 0.22) attenuation-compensation. (E to H) Light-sheet cross sections resulting from digital scanning of the Bessel beams shown in (A) to (D). (I and J) Peak transverse intensity as a function of longitudinal coordinate for the Bessel beams (A to D) and Bessel light sheets (E to H), respectively. The intensity is normalized to the start of the propagation-invariant region (x = −57.5 μm). (K to T) Comparison of Bessel beam and light-sheet transverse profiles with and without attenuation-compensation at x = −50 μm (K and P), −25 μm (L and Q), 0 μm (M and R), 25 μm (N and S), and 50 μm (O and T). (U and V) RMSE between Bessel beam and light-sheet transverse profiles with and without attenuation-compensation as a function of longitudinal coordinate.

  • Fig. 3 Attenuation-compensated Airy LSM in an attenuating phantom.

    Maximum intensity projections of recorded data (A, E, and I) and deconvolved images (B, F, and J) of sub–diffraction-limited fluorescent microspheres in an absorbing phantom with Cattn = 55 ± 1 cm−1. (A and B) No attenuation-compensation (σ = 0), (E and F) σ = 0.23, and (I and J) σ = 0.46 (full attenuation-compensation). (C, D, G, H, K, and L) Zoomed-in views of the regions indicated by the dashed boxes (i) and (ii) in (B), (F), and (J). (M and N) Axial resolution determined by FWHM of fluorescent microspheres and local SBR, respectively, as a function of light-sheet propagation (mean ± SD, 10-μm binning); σ = 0 (blue), σ = 0.23 (green), and σ = 0.46 (red). (O and P) Ratios of the graphs shown in (M) and (N); σ = 0.23/σ = 0 (cyan) and σ = 0.46/σ = 0 (magenta). Look-up tables of the images shown in (A) to (L) are independently scaled to the data shown.

  • Fig. 4 Attenuation-compensated Airy LSM in S. lamarcki opercula.

    Maximum intensity projections of deconvolved Airy LSM images of nuclei stained with propidium iodide (PI) in the operculum of S. lamarcki (attenuation estimated at 85 cm−1) with (A) no attenuation-compensation, (B) σ = 0.23, and (C) σ = 0.46. (D to F) Expanded views of the region indicated by the dashed box in (A) to (C). (G to I) Intensity profiles along the dashed line shown in (D) to (F). Line intensity profiles are shown relative to the noise floor, given by the local mean background μb.

  • Fig. 5 Attenuation-compensated Airy LSM in S. lamarcki opercula.

    Maximum intensity projections of deconvolved Airy LSM images of nuclei stained with PI in the operculum of S. lamarcki (attenuation estimated at 75 cm−1) with (A) no attenuation-compensation, (B) no attenuation-compensation but the same total power as a compensated light sheet (σ = 0.46), and (C) σ = 0.46. (D to G) Zoomed-in views of the region indicated by dashed boxes (i) to (iv) in (A). (H to O) Same regions from (B) and (C). Intensity profiles along the dashed line in (F), (J), and (N) are shown in (P) to (R), respectively. Line intensity profiles are shown relative to the noise floor, given by the local mean background μb.

  • Fig. 6 Attenuation-compensated Airy LSM in mouse brain section.

    Maximum intensity projections of deconvolved Airy LSM images of kisspeptin neurons expressing mCherry in the hypothalamic arcuate nucleus of a mouse brain (attenuation estimated at 100 cm−1) with (A) no attenuation-compensation and (B) σ = 0.54. (C and D) Expanded views of the region indicated by the dashed box in (A) and (B). (E and F) Orthogonal projections of the regions shown in (C) and (D). Filled arrowheads indicate the positions of dendritic spines in (C) and (D). Dashed arrowheads in (C) indicate the position of dendritic spines not identified without attenuation-compensation.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/eaar4817/DC1

    note S1. Attenuation-compensation of an Airy beam light sheet

    note S2. Attenuation-compensation of a Bessel beam light sheet

    note S3. Modification of deconvolution protocol incorporating attenuation and attenuation-compensation

    note S4. Effect of incorrect attenuation estimation on deconvolution

    note S5. Determination of specimen attenuation

    note S6. Sample-based geometric effects on attenuation

    note S7. Attenuation-compensation of multiphoton excitation Airy and Bessel light sheets

    fig. S1. Pupil functions of attenuation-compensated Airy and Bessel beams.

    fig. S2. Look-up tables of achievable attenuation-compensation for Airy and Bessel beam parameters.

    fig. S3. Line profiles through simulated images of a 1D resolution target shown in Fig. 1 (main text).

    fig. S4. Simulated images: Effect on deconvolution from error in estimation of attenuation.

    fig. S5. Sample induced geometric effects on attenuation profile across the FOV.

    fig. S6. Attenuation-compensated LSM in a scattering sample.

    fig. S7. Local SBR and CNR measured for data shown in Fig. 4 (main text).

    fig. S8. Local SBR and CNR measured for data shown in Fig. 5 (main text).

    fig. S9. Light-sheet intensity profiles for the data shown in Fig. 5 (main text).

    fig. S10. Effect of attenuation and attenuation-compensation on two-photon excitation SPIM and DLSM Airy light sheet.

    fig. S11. Effect of attenuation and attenuation-compensation on two-photon excitation Bessel beam.

    fig. S12. Effect of attenuation and attenuation-compensation on two-photon excitation DSLM Bessel light sheet.

    fig. S13. Schematic of attenuation-compensated Airy light-sheet microscope.

    table S1. Experimental parameters for all data shown in main text.

    table S2. FOV of two-photon excitation SPIM and DLSM Airy light sheet with attenuation and attenuation-compensation.

    table S3. FOV of two-photon excitation Bessel beam and DLSM Bessel light sheet with attenuation and attenuation-compensation.

  • Supplementary Materials

    This PDF file includes:

    • note S1. Attenuation-compensation of an Airy beam light sheet
    • note S2. Attenuation-compensation of a Bessel beam light sheet
    • note S3. Modification of deconvolution protocol incorporating attenuation and attenuation-compensation
    • note S4. Effect of incorrect attenuation estimation on deconvolution
    • note S5. Determination of specimen attenuation
    • note S6. Sample-based geometric effects on attenuation
    • note S7. Attenuation-compensation of multiphoton excitation Airy and Bessel light sheets
    • fig. S1. Pupil functions of attenuation-compensated Airy and Bessel beams.
    • fig. S2. Look-up tables of achievable attenuation-compensation for Airy and Bessel beam parameters.
    • fig. S3. Line profiles through simulated images of a 1D resolution target shown in Fig. 1 (main text).
    • fig. S4. Simulated images: Effect on deconvolution from error in estimation of attenuation.
    • fig. S5. Sample induced geometric effects on attenuation profile across the FOV.
    • fig. S6. Attenuation-compensated LSM in a scattering sample.
    • fig. S7. Local SBR and CNR measured for data shown in Fig. 4 (main text).
    • fig. S8. Local SBR and CNR measured for data shown in Fig. 5 (main text).
    • fig. S9. Light-sheet intensity profiles for the data shown in Fig. 5 (main text).
    • fig. S10. Effect of attenuation and attenuation-compensation on two-photon excitation SPIM and DLSM Airy light sheet.
    • fig. S11. Effect of attenuation and attenuation-compensation on two-photon excitation Bessel beam.
    • fig. S12. Effect of attenuation and attenuation-compensation on two-photon excitation DSLM Bessel light sheet.
    • fig. S13. Schematic of attenuation-compensated Airy light-sheet microscope.
    • table S1. Experimental parameters for all data shown in main text.
    • table S2. FOV of two-photon excitation SPIM and DLSM Airy light sheet with attenuation and attenuation-compensation.
    • table S3. FOV of two-photon excitation Bessel beam and DLSM Bessel light sheet with attenuation and attenuation-compensation.

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