Fig 1.
(A) Schematic of the instrument design, with labels corresponding to the parts list in S1 Table. See also S1 Movie. The excitation laser line is selected by an acousto-optic tunable filter (AOTF2), then directed to a galvanometer mirror (G3) and objective lens (L4) to create a time-averaged sheet of light in the sample chamber (C) via a prism (Pr5). Specimens flow through a system of tubing controlled by a syringe pump (Pu9) and valves (V10) and are automatically positioned in a square-walled capillary (Cap11) for imaging. Bright field images are used for positioning the sample and are illuminated with an LED (LED6). After imaging, specimens are directed into a reservoir (R). (B) Schematic of the imaging area. The 3D-printed sample chamber (C), prism (Pr5), and imaging capillary (Cap11) are apparent. (C) Photograph of the imaging area corresponding to the schematic in (B).
Fig 2.
Specimen positioning and image quality.
(A) Composite brightfield image of a larval zebrafish positioned in a glass capillary. Scale bar: 500 μm. (B) Normalized intensity averaged along the short axis of the brightfield image, and the intensity of the template image that best matches the fish in (A). Cross-correlation with the template is used to automatically position the fish for light sheet fluorescence imaging. (C) Light sheet fluorescence images of a 28 nm diameter fluorescent microsphere, showing x-y and z-y planes centered on the particle. (D) Line-scan of intensity along the detection axis (z) through a fluorescent microsphere, with a Gaussian fit showing a width of approximately 3 μm.
Fig 3.
Imaging neutrophils in larval zebrafish.
(A) A maximum intensity projection of a three-dimensional light sheet fluorescence image of GFP-expressing neutrophils near the intestine of a 5 dpf larval zebrafish. The 3D scan is provided as S2 Movie. The intestine and swim bladder are roughly outlined by the yellow dotted lines. Scale bar: 100 μm. (B) The total number of neutrophils in each fish; the x-axis is ordered by neutrophil count. (C) Neutrophil count along the anterior-posterior dimension, summed over all fish examined (N = 41). The x-axis corresponds approximately to the horizontal range of (A). (D) The total intensity of the detected neutrohils per fish vs the total number of neutrophils in that fish. The two measures are weakly correlated with a coefficient of determination R2 = 0.4.
Fig 4.
Neutrophil counts after exposure to LPS.
The number of neutrophils counted from light sheet fluorescence images of larval zebrafish after exposure to 150 μg/ml LPS for 0 (control), 2, or 24 hours. At 2 hours post-exposure, there was no discernible difference between the LPS-treated group and the control group. At 24 hours post-exposure, the LPS group displayed an increase in mean neutrophil count of 1.2 ± 0.1.