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For: Thin-sheet laser imaging microscopy for optical sectioning of thick tissues
This file can be viewed as an interactive 3-D PDF file in Adobe Acrobat 9 (Adobe Systems Incorporated, San Jose, CA, USA). Dual-laser illuminators are positioned along an x axis optical bench rail with the specimen chamber positioned between the illuminators. The specimen is moved in the x, y, and z directions by three micropositioners and by a rotating stage which is attached to the specimen rod that extends into the fluid filled chamber.
A movie showing a stack of 134 1-μm serial sections of the scala media of the adult mouse cochlea that has been virtually resectioned in an oblique plane along the outer hair cells.
Supplementary Movie 2. TSLIM optical sections of the spiral ganglion. A movie showing a stack of 82 1-μm serial sections through the spiral ganglion of the mouse cochlea showing neuron cell bodies, nuclei, and their axons.
A movie showing a stack of 100, 20 μm serial transverse sections through the head of a Casper zebrafish showing the brain and a 3D reconstruction of he inner ear.
Tissues from the mouse, zebrafish, and rat were used to illustrate some of the capabilities of TSLIM. However, TSLIM can be used for optical sectioning of many different types of tissues and organisms. Since TSLIM is modular, it can be configured with different lasers, beam expanders, lenses, and specimen chambers for different types of specimens. For high resolution of small structures, a thin beam waist is required and TSLIM could be configured for single-beam, full-frame imaging. Exposure times varied depending upon tissue fluorescence, specimen thickness, the number of optical sections desired, and whether single- or dual-beam illumination was used. A typical length of time to obtain a single, full-frame optical section using dual-beam illumination was 1 s. However, for high-resolution structures in a large specimen, a full-frame image would appear focused only within the confocal region of the light sheet. This is shown in Figure 2 using the mouse cochlea and zebrafish head. In Figure 2A and 2C the arrow indicates the approximate position of the beam waist and in that region the specimen appears well focused. To the left and right of the beam waist, the optical section appears out of focus due to increasing thickness of the light sheet away from the focal point of the lens. To provide optimal resolution and focus across the full width of a specimen, the specimen was moved across the beam waist of the light sheet and image columns (the size of the confocal region) were obtained and stitched together to form a well focused, composite image (Figure 2, B and D). It took 46 s and 74 s to produce the composite image in Figure 2, B and D, respectively. In addition, horizontal lines are noticeable in the left portion of Figure 2A: these are produced by the uneven absorption of light by certain tissue structures as the light passes through the specimen. These absorption artifacts, which are common in light-sheet microscopy, are minimized by dual-beam illumination in TSLIM (7, 11). Huisken et al. (11) also used beam oscillation to reduce absorption lines, but their method required complex equipment and software.
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