Research ArticleMICROSCOPY

3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

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Science Advances  11 Aug 2017:
Vol. 3, no. 8, e1700553
DOI: 10.1126/sciadv.1700553
  • Fig. 1 Lens-free on-chip microscopy setup and image processing steps.

    (A) Schematic of the lens-free on-chip imaging setup. The cleared tissue is loaded in a polydimethylsiloxane (PDMS)/glass chamber filled with a refractive index matching solution. A sealant is applied on the sides to avoid evaporation and leakage. RIMS, refractive index matching solution; CMOS, complementary metal-oxide semiconductor. (B) Lens-free image processing workflow is outlined.

  • Fig. 2 Imaging comparison of different thicknesses of cleared tissue.

    (A to C) Sub-FOVs of the pseudocolored lens-free reconstructed images of a 50-, 100-, and 200-μm-thick cleared mouse brain tissue. Each one of these images was digitally focused to a few arbitrary cells located within the sample. The 50-μm-thick sample is the same sample shown in fig. S1. (D to F) Nine randomly selected cells from each sample thickness are illustrated. (G) Mean CNR of the reconstructed neurons within the cleared tissue as a function of its thickness. Error bars represent the SEM, which is equal to the SD divided by the square root of the number of sampled cells.

  • Fig. 3 Optimization of pH for tissue staining.

    (A) Randomly selected cells that are reconstructed using lens-free on-chip microscopy corresponding to four cleared tissue samples (each 200 μm thick) stained with pH values of 6.7, 6.9, 7.1, and 7.4. Scale bar, 10 μm. (B) Average CNR as a function of the pH value, with the peak CNR occurring at a pH value of 7.1. Error bars represent the SEM.

  • Fig. 4 Effect of the number of heights and the sparsity-based image denoising algorithm on the CNR of the reconstructed lens-free images corresponding to a 200-μm-thick cleared tissue sample stained under a pH of 7.1.

    (A and B) Sample lens-free images of three randomly selected cells as the number of heights varies from 2 to 8, before and after applying the sparsity constraint, respectively. (C) Average CNR calculated using the reconstructed lens-free images of 27 cells plotted against the number of heights, before (black curve) and after (red curve) applying the sparsity constraint. Error bars represent the SEM. Using the sparsity constraint significantly improves the CNR of the lens-free images.

  • Fig. 5 Lens-free 3D imaging of a cleared, DAB-stained, 200-μm-thick mouse brain tissue.

    (A) Full FOV lens-free hologram. (B) A zoomed-in region corresponding to a 20× microscope objective FOV. MIP images of the lens-free pseudocolored z-stack and the scanning microscope’s z-stack [obtained with a 20× objective (NA = 0.75)] are presented. (C) Comparison of lens-free images of 19 neurons against the images obtained with a 20× objective lens (NA = 0.75).

  • Table 1 Data and timing efficiency.

    Left: Comparison of the number of images and the amount of acquired image data between a lens-free on-chip microscope and a typical scanning optical microscope with a 20× objective lens. Right: Computation time corresponding to the full FOV (20.5 mm2) image reconstruction routine implemented in CUDA using an Nvidia Tesla K20c graphics processing unit (GPU) (released in November 2012). This total computation time can be further improved by more than an order of magnitude by using a GPU cluster.

    Data efficiency comparisonTiming of lens-free image reconstruction
    Lens-free microscopeSubroutineTime (s)
    HDRPSRHeightsNo. of imagesData (GB)Read images from hard drive7.0
    33633243.5Autofocus10.2
    Conventional scanning microscope 20× (NA = 0.5)PSR91.0
    Lateral scanAxial scanNo. of imagesData (GB)Image alignment54.6
    9273671639.4Multi-height phase recovery94.6
    Total257.4

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700553/DC1

    fig. S1. Illumination wavelength optimization using a 50-μm-thick tissue sample.

    movie S1. Side-by-side comparison of a lens-free 3D z-stack of a CLARITY-cleared, DAB-stained 200-μm-thick mouse brain tissue with that of a scanning optical microscope using a 20× objective lens (NA = 0.75).

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Illumination wavelength optimization using a 50-μm-thick tissue sample.
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Side-by-side comparison of a lens-free 3D z-stack of a CLARITY-cleared, DAB-stained 200-μm-thick mouse brain tissue with that of a scanning optical microscope using a 20× objective lens (NA = 0.75).

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