Mechanically sheared axially swept light-sheet microscopy

Abstract

We present a mechanically sheared image acquisition format for upright and open-top light-sheet microscopes that automatically places data in its proper spatial context. This approach, which reduces computational post-processing and eliminates unnecessary interpolation or duplication of the data, is demonstrated on an upright variant of axially swept light-sheet microscopy (ASLM) that achieves a field of view, measuring 774 × 435 microns, that is 3.2-fold larger than previous models and a raw and isotropic resolution of ∼460 nm. Combined, we demonstrate the power of this approach by imaging sub-diffraction beads, cleared biological tissues, and expanded specimens.

Fig. 1.

Optical and mechanical shearing of imaging data. (a) In a conventional oblique scan format, the sample is scanned in the S-direction and images are acquired at each adjacent plane in a staggered format (top) but saved in a continuous format (middle). Thus, data must be computationally sheared (bottom) to place it back into its proper spatial context, which introduces empty space above and below the shear axis (see black outline around sheared image). (b) In a mechanically sheared oblique scan format, the sample is simultaneously scanned in S and Z’, thereby placing it in its proper spatial context during image acquisition.

Fig. 2.

Analysis of 200 nm beads. (a) Displays the XY maximum intensity projection of 200 nm beads in agarose spanning a 20 µm range in the Z dimension. (b) Shows zoomed-in regions of the image from panel a, arranged from left to right. (c) Depicts the XZ maximum intensity projection of 200 nm beads in agarose across a 20 µm range in the Z dimension. (d) Exhibits zoomed-in regions of the image from panel c. (e) Illustrates the maximum intensity projection of a single 200 nm bead in the XY, XZ, and YZ dimensions. (f) Presents histograms of the full-width half maximum (FWHM) of 200 nm beads in the X, Y, and Z dimensions. The number of analyzed beads is 713. (g) Shows a heatmap of the lateral full-width half maximum (FWHM) of 200 nm beads across a 435 µm x 774 µm camera chip. Scale bars: a, c = 100 µm; b, d = 10 µm; e = 1 µm.

Fig. 3.

Analysis of 200 nm beads for mechanically and computationally sheared data sets. The mean resolution for mechanically sheared data was 491 nm (X), 477 nm (Y), and 632 nm (Z), whereas for computationally sheared data, it was 473 nm (X), 468 nm (Y), and 723 nm (Z), respectively. Statistical significance was evaluated with a Mann-Whitney U test, which makes no assumptions about the underlying population statistics. P-values were 0.003, < 0.0001, and <0.0001 in X, Y, and Z, respectively (see Supplement 1 Table S1). All statistical tests were performed with the SciPy toolkit [20].

Fig. 4.

BABB cleared human kidney imaged with mechanical shearing. Specimen was labeled with FLARE [22], and carbohydrates are shown as blue, and proteins are shown as red. (a) Maximum intensity projection of a human nephron. (b) Zoom in a single slice of the region highlighted in image (a). Glomerulus and red blood cells in 3 different dimensions. (c) A volume rendering of glomerulus and red blood cells from (b). Scale bars: a = 100 µm; b, c = 20 µm.

Fig. 5.

Protein retention expansion microscopy of mouse liver tissues. (a) A three-dimensional volume rendering showcasing mechanically sheared ASLM images of expanded mouse liver tissue exhibiting melanoma micro-metastases. The rendering displays a volume measuring 774.14 × 418.66 × 100 µm. (b) Orthogonal planes from a 287.28 × 261.07 × 100 µm isotropic volume, provide a detailed view of the micro-metastasis. The imaging includes gray for nuclear structures, green for Myosin IIa, and magenta for amines. (c) A 3D projection of the nuclear channel, oriented at a 45-degree angle. (d) A focused view on the melanoma micro-metastasis area of c. (e) Visualization of nuclei with surrounding Myosin IIa signaling in the micro-metastasis region. (f) A merged view incorporating all three channels to illustrate the micro-metastasis area. (g) Volume rendering of M-shearing ALSM images of healthy mouse liver tissue, demonstrating: Gray for nuclear structures and green for Collagen I. (h) A maximum intensity projection offering high-resolution imaging of the sample. (i) Orthogonal planes from the region indicated in h, providing an enhanced view. All images are accompanied by a scale bar measuring 100 µm. The expansion factor was ∼ 4.5 for all samples.

Fig. 6.

Imaging of expanded, ∼28 × 20 × 0.25 mm human colon specimen. (a) Low-resolution overview image of FLARE-stained and expanded tissue specimen. Image acquired on a widefield microscope at 10X magnification and transferred to the ASLM system. (b-g) Local high-resolution images acquired from regions shown in (a), presented as single 2D cross-section in X and Y. Scale bar: a = 5 mm; b-g = 100 µm.

Fig. 7.

Large volume imaging of expanded human colon specimen. (a) A ∼2.8 × 3.5 × 0.2 mm volume imaged in a mechanically sheared and tiled format. Specimen was stained using FLARE and is presented as a slice after 4X down sampling. (b-c) High-resolution images of sub-regions from (a). (d) Ortho-slice of region shown in (a). Scale bar: a = 200 µm; b, c, d = 100 µm.