Figure 1.

The optical resolution of light-sheet microscopy is determined by both detection objective NA and thickness of the illuminating laser sheet. In conventional light-sheet microscopy, there is a fundamental tradeoff between light-sheet thickness and the field-of-view; when a thin laser sheet is illuminated (hence a higher axial resolution), the effective imaging area (determined by Rayleigh length) must decrease rapidly. To overcome this tradeoff, one approach is to use a beam that is generated by a sequence of aspheric lenses (Saghafi et al., 2014). A, The optical design of this light-sheet generator, along with the measurements of the beam profile along the x-y plane for confirmation of the extended Rayleigh range. Each optical element in the design are as follows: (a) first aspheric condenser lens, (b) Powell lens, (c) second aspheric condenser lens, (d) first achromatic cylindrical lens, and (e) second achromatic cylindrical lens. B, A Scheme of the ultramicroscopy setup: (1) 488 nm laser; (2) light-sheet generator optics, duplicate arms are placed on the left and right side of the specimen container; (3) specimen container with quartz windows; (4) detection arm with an exchangeable objective; and (5) scientific grade CCD camera. The objective for imaging is dipped into the clearing solution from above so that the objectives can be exchanged easily for change of magnification. C–E, Using a custom-made ultramicroscope equipped with the novel light-sheet generator, a cleared fruit-fly brain was imaged and reconstructed in 3D. A virtual cross-section through the head is presented in D. om, Ommatidia; m, medulla; lo, lobula. A virtual cross-section through the thorax is presented in E. oe, Esophagus; pv, proventriculus (cardia). F, G, The hippocampus area of Thy1-EGFP-M mouse brain was dissected and cleared, and imaged with ultramicroscopy. Close-up with 10× magnification (F) and 20× magnification (G) are shown. Scale bars: F, G, 100 μm .