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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Curr Protoc Cell Biol. 2018 Aug 2;80(1):e56. doi: 10.1002/cpcb.56

Figure 11.

Figure 11

Imaging expanded ExM samples with light sheet microscopes. (A) Schematics showing configurations of the objectives and the mounted sample on a commercial light sheet microscope (Zeiss Z.1 light sheet). The green and red arrows show the excitation and emission light, respectively. Note that the gel is facing the objective and is mounted on a coverslip as the backing, which is glued onto a 3D-printed adapter that is connected to the rod-shaped sample holder hanging from the top (the rod is not shown in the schematic). The sample is immersed in water or diluted buffer and the excitation light sheet travels through the sides of the gel perpendicular to the detection objective focal axis. (B) Sample mounting inside the Zeiss Z.1 light sheet microscope, with the imaging chamber removed. Two illumination objectives are on the side to form the excitation light sheets, and a water-immersion objective is located perpendicular to the illumination objectives for the imaging. (C) The same setup as B with the imaging chamber installed. The two side glass windows allow the light sheet to illuminate the sample and the front glass window is used for rough sample alignment using a small camera. Together with the imaging objective at the back, the chamber is sealed and is filled with water (or diluted PBS for ExFISH samples) during the imaging. (D) 3D representation of Zeiss Z.1 light sheet microscopy image of Thy1-YFP (green) brain tissue with ExFISH targeting YFP (red) and Gad1 (blue) mRNAs. (E) Maximum intensity projection (~8 μm in Z) of a subvolume of D demonstrating the capability to resolve single RNA molecules within a cell body after expansion. (F) Volume rendering of a subvolume of D. Scale bars, 10 μm (E, in pre-expansion units) and 20 μm (F, in pre-expansion units). About 3x expansion factor for all samples. Adapted from (Chen et al., 2016). (G) Rendered image of a section of a large scale 3-D multicolor recording of a virally delivered Brainbow3.0 mouse hippocampus section using pre-expansion staining proExM, and imaged on a Zeiss Z.1 light sheet microscope. The expansion factor is ~4.5x and the resulting effective lateral pixel size is 60 nm. Scale bar: 100 μm (in pre-expansion units).