Article Figures & Data
Figures
- Figure 1.
Shadowing with Gaussian and Bessel beams. A, Generation of shadows when focusing a Gaussian beam with a microscope objective (Ex). Fluorescent particles positioned in the elongated shadow cannot be excited. B, For Bessel beams, here generated by a Gaussian beam impinging on a conical lens called axicon, the optical power stored in the concentric rings can regenerate the initial beam profile in the reconstruction region behind the shadow zone. Fluorescent particles behind the conical shadow can be excited and therefore imaged.
- Figure 2.
Generation of a Bessel beam using an axicon lens. The characteristic transverse J0 Bessel beam profile of a central lobe with concentric rings is created within the propagation length δ Z of the axicon which is a function of the input beam radius d and the angle α of the conical lens.
- Figure 3.
A, The custom-made LSM uses double sided illumination by either Gaussian or Bessel beams. B, Measured PSFs for the lateral and axial direction for Gaussian (red) and Bessel illumination (cyan) using fluorescent beads. The FWHMs of the PSF are reported in the table for Gaussian and Bessel illumination, respectively. C, PSFs for Gaussian and Bessel illumination. D, Longitudinal beam profile. E, Transversal profile for the Gaussian (red) and Bessel beam (cyan). F, Beam width ω(z) of the Gaussian beam extracted from profile shown in F. Red line indicates fit to hyperbolic function. The beam waist ω0, the Rayleigh range ZR, and the beam NA were extracted from the fit. Bottom, Table of all beam parameters. * indicates theoretically derived values.
- Figure 4.
Striping with Gaussian illumination and estimation of the volume affected by severe streaking parallel to the illumination direction (yellow arrows). A, Axial sections through the brain of a FosTRAP mouse. Scale bar: 1 mm. Inset, top, Striping caused by absorbing structures on the surface (red arrowhead). Bottom, Striping caused toward the center line of the brain by progressive absorption. B, Axial sections through the brain of a PV-tdTomato mouse. Scale bar: 1 mm. Inset, top, Striping caused by a absorption of a particularly bright part of the olfactory bulb (red arrowhead). Bottom, Shadow caused by an internal structure, presumable due to incomplete optical clarification (red arrow). C, Axial sections through the brain of a FosTRAP mouse. Scale bar: 1 mm. Inset, Striping caused by bubbles settling on the brain surface (red arrows). The same bubbles cause the circular shadows on the detection path (red arrow heads). D, Percentage of the brain volume affected by streaking in n = 8446 stitched whole-brain images from N = 10 animals. Line is fit to Gaussian. E, Variation of striping within axial sections throughout the depth of one mouse brain (shown in C).
- Figure 5.
Structural neuron imaging. A, Maximum intensity projection over 20 µm of a Thy1-GFP-M axial mouse brain section imaged with a Gaussian and a Bessel beam (B). Yellow arrows indicate direction of light-sheet propagation. Each half of the brain was excited by one light sheet, respectively. White box marks position of details in the hippocampus affected by streaking artifacts for Gaussian and Bessel beam illumination, respectively (insets). Scale bar: 10 µm. C, Line profile averaged over the entire height of the inset evidences the shadows as drops in the red curve. D, Sensitivity of striping to chosen threshold. E, Calculating the absolute value of the difference in intensity line profile between Gauss and Bessel allows to estimate the area affected by streaking artifacts by applying a threshold (here 5%). Applying this threshold to stitched images of half a brain (F, bottom row) over a depth of 400 µm with a step size of 2 µm yielded that 37.5 ± 3.1% (error is SD) of the dataset was affected by streaking artifacts.
- Figure 6.
Vascular structural imaging. A, Adult Thy1-GFP-M mouse with photothrombotic stroke in the primary motor cortex and brain vasculature labeling with tetramethylrhodamine-albumin imaged with Gaussian illumination. Yellow arrows indicate light-sheet propagation. White box in the olfactory bulb corresponds to images B–E. Strong shadows obscure even large vessels when illuminated with Gaussian (B) but not with Bessel beam illumination (C). Scale bar: 10 µm. Automated segmentation based on simple thresholding for Gaussian (D) and Bessel beam illumination (E). F, G, Isometric views of red (cyan) box in B, C shown along yz and xz for Gaussian (Bessel) illumination. H, I, 3D projections of the segmented data corresponding to the red and cyan ROIs indicated in B, C. J, Manders coefficients averaged over a 400-µm stack comprising images in D, E for Gaussian and Bessel illumination. The fraction of total intensity in the Bessel channel located in pixels of non-zero intensity in the Gaussian channel was 0.62 ± 0.02, whereas the corresponding value for the Gaussian channel was 0.87 ± 0.01 (p < 0.0001, paired t test, n = 39, error is SEM). See also Movies 1–8 for this figure. ****p ≤ 0.0001.
Movies
- Movie 1.
Raw data of the vasculature of a Thy1-GFP-M mouse labelled with tetramethylrhodamine-albumin imaged with Gaussian illumination. See Figure 6B for details.
- Movie 2.
Raw data of the vasculature of a Thy1-GFP-M mouse labelled with tetramethylrhodamine-albumin imaged with Bessel beam illumination. See Figure 6C for details.
- Movie 3.
Segmented data of the vasculature of a Thy1-GFP-M mouse labelled with tetramethylrhodamine-albumin imaged with Gaussian illumination. Automated segmentation was based on simple thresholding. See Figure 6D for details.
- Movie 4.
GFP-M mouse labelled with tetramethylrhodamine-albumin imaged with Bessel beam illumination. Automated segmentation was based on simple thresholding. See Figure 6E for details.
- Movie 5.
3D projection of raw data of the vasculature of a Thy1-GFP-M mouse labelled with tetramethylrhodaminealbumin imaged with Gaussian beam illumination. A look-up table was applied for clarity. See Figure 6F for details.
- Movie 6.
3D projection of raw data of the vasculature of a Thy1-GFP-M mouse labelled with tetramethylrhodaminealbumin imaged with Bessel beam illumination. A look-up table was applied for clarity. See Figure 6G for details.
- Movie 7.
3D projection of segmented data of the vasculature of a Thy1-GFP-M mouse labelled with tetramethylrhodaminealbumin imaged with Gaussian beam illumination. Automated segmentation was based on simple thresholding. See Figure 6H for details.
- Movie 8.
3D projection of segmented data of the vasculature of a Thy1-GFP-M mouse labelled with tetramethylrhodaminealbumin imaged with Bessel beam illumination. Automated segmentation was based on simple thresholding. See Figure 6I for details.
In this issue
