Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection

Abstract

It is well known that light-sheet illumination can enable optically sectioned wide-field imaging of macroscopic samples. However, the optical sectioning capacity of a light-sheet macroscope is undermined by sample-induced scattering or aberrations that broaden the thickness of the sheet illumination. We present a technique to enhance the optical sectioning capacity of a scanning light-sheet microscope by out-of-focus background rejection. The technique, called HiLo microscopy, makes use of two images sequentially acquired with uniform and structured sheet illumination. An optically sectioned image is then synthesized by fusing high and low spatial frequency information from both images. The benefits of combining light-sheet macroscopy and HiLo background rejection are demonstrated in optically cleared whole mouse brain samples, using both green fluorescent protein (GFP)-fluorescence and dark-field scattered light contrast.

Figure 1

Layout of scanning light-sheet microscope. The laser beam power is modulated with an acousto-optic modulator (AOM) and expanded with a 2 to 5×beam expander. The beam is then focused into a sample with an f=150-mm lens. The sample is mounted on a motorized rotary stage, which is itself mounted on a platform whose height is controlled by a motorized linear translation stage. Fast horizontal scanning of the laser beam is performed with a piezomounted tilt mirror to produce a light sheet. Uniform and structured illumination images are acquired during scan fly-forward and fly-back, respectively. Slow vertical light-sheet tracking is ensured with a motorized tilt mirror. The setup is displayed as a top view. The inset in dashed lines is a front view.

Figure 2

Scanning light-sheet images of an optically cleared whole mouse brain (P14) whose hippocampal pyramidal neurons have been targeted with EGFP via in utero electroporation (Cb: cerebellum, Cx: cortex, Hi: hippocampus, Mid: midbrain, Str: striatum, CA1∕3: area of Ammon’s horn, DG: dentate gyrus). Top panels are images acquired with (a) uniform and (b) structured sheet illumination. (c) is the resultant HiLo processed image. Sparsely labeled neurons stand out as bright fluorescent objects amid lower level autofluorescence. (d), (e), and (f) are zoomed maximum intensity projections (54-μm depth range) of a single cell. Fields of view are 8 mm (top) and 2.3 mm (bottom). Laser power was ∼1 mW. Camera exposure time per image was 250 ms.

Figure 3

Scanning light-sheet images of the half forebrain region of an optically cleared whole mouse brain of older age P45 (Cx: cortex, Mid: midbrain, Str: striatum). Images are acquired in a dark-field scattering mode. A network of myelinated axon processes in the alveus (Alv) of the hippocampus and striatum is clearly visible. Grayscale panels are average intensity projections (∼100-μm depth range) of (a) uniform and (b) structured illumination images, and (c) the corresponding HiLo images. Field of view is 4.8 mm. Laser power was ∼0.15 mW (no emission filter). Camera exposure time per image was 350 ms.

Figure 4

Same data as in Fig. 3. The color image represents an overlay of a single frame from the uniform illumination (red) and HiLo (cyan) stacks. Pixel values along a horizontal line (green) are shown in the plot, depicting uniform illumination (red) and HiLo (black) traces. Both traces are normalized to their respective maximum values.

Figure 5

Combined fluorescence (green) and dark-field (red) HiLo images of the hippocampus region of a whole thy-1 mouse brain (same sample as in Fig. 3). (a), (b), and (c) are frames acquired from various depths within an image stack. The field of view is 4.8 mm. The laser power was 1 and 0.15 mW for fluorescence and dark-field imaging, respectively. The camera exposure time was 350 ms per frame.

Video 1

Maximum intensity projection of the entire stack of combined fluorescence (green) and dark-field (red) HiLo images of the hippocampus region of a whole thy-1 mouse brain (same data as in Fig. 5). The stack spans a 1-mm depth range with an interframe separation of 10 μm, and is presented as a 3-D projection (MP4, 1 MG). .