Abstract
We describe a water-based optical clearing agent, SeeDB (See Deep Brain), which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. SeeDB is a saturated solution of fructose (80.2% w/w) in water with 0.5% α-thioglycerol. In standard SeeDB optical clearing procedure, we treat paraformaldehyde-fixed embryo and brain samples with increasing concentrations of aqueous fructose solutions, and finally equilibrate them in SeeDB. The entire procedure takes approximately three days. Unlike previous methods, this method maintains a constant sample volume during the clearing procedure, an important factor to keep cellular morphology intact. After optical clearing, we can reach > 1,000 μm under confocal microscopy. When combined with two-photon microscopy, SeeDB allows us to image fixed mouse brains at millimeters-scale level. This method facilitates comprehensive and quantitative analyses for understanding neuronal circuitry, both in the adult and developing mouse brain. A SeeDB variant (SeeDB37) and optimized procedures (SeeDBp and SeeDB37ht protocols) are also supplied for specific requirements.
Keywords: Tissue clearing, Fluorescence imaging, Connectome, SeeDB, Fructose
Materials and Reagents
Equipment
Procedure
Part I. Choice of protocols
Part II. Choice of objective lenses and imaging Because refractive index of SeeDB is 1.49, it is important to choose appropriate objective lenses to minimize spherical aberration. In the two-photon microscopy, index-matched and long working distance customized objective lens is most ideal to obtain high-resolution images at millimeter-scale. We used custom-made objective lens from Olympus (it will be commercialized from Olympus). Commercial water or Scale immersion objective lens can be used for imaging up to 2-4 mm depth. In the confocal microscopy, a water-immersion objective lens (refractive index 1.33) works better than an air-immersion objective lens (refractive index 1.0) (Figure 5). Glycerol/Oil immersion lenses are also suitable. Customized objective lens is not necessary for confocal imaging; we did not see obvious differences in resolution within 1 mm depth. When using an air or water-immersion lens, the imaging depth should be calibrated because an apparent axial scale is shortened in the SeeDB solution due to its high refractive index (refractive index 1.49).
Anticipated Results
Figure 4. Transmittance images of neonatal, P21, and adult mouse brain samples. Transparency can be evaluated by eye under the light source. Typically, adult samples are more difficult to clear than young samples.
Figure 5. Confocal imaging of adult brain slices (Thy1-YFP-H mouse). Water-immersion objective lenses work better than air lenses because of smaller spherical aberration in SeeDB. The maximum imaging depth possible is ~500 μm at spine resolution, ~1 mm at fiber resolution, and ~2 mm at cellular resolution. In the two-photon microscopy, the maximum imaging depth possible with commercial 25x objective lenses is ~1 mm at spine resolution and 3-4 mm at fiber resolution. When a commercial water- or air-immersion objective lens is used for imaging cleared samples, depth (z) needs to be calibrated. In order to obtain the correct z position in the sample, the real depth is calculated by multiplying the depth by nSeeDB and then dividing by nobjective, where n represents refractive index. nSeeDB = 1.49; nSeeDB37 = 1.50; nair = 1.0; nH2O = 1.33.
Recipes
Troubleshooting
Acknowledgments
This protocol was adapted from our original publication of the protocol (Ke et al., 2013). We thank J.R. Sanes for providing mouse strains. This work was supported by grants from the PRESTO program of the Japan Science and Technology Agency, the Sumitomo Foundation, the Nakajima Foundation, the Mitsubishi Foundation, the Strategic Programs for R&D (President's Discretionary Fund) of RIKEN, and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (KAKENHI 23680038). The imaging experiments were supported by the RIKEN Center for Developmental Biology Imaging Facility. Animal experiments were supported by the Laboratory for Animal Resources and Genetic Engineering at the RIKEN Center for Developmental Biology.
References
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Hi, Anirudh,Please find the SeeDB recipes in Recipes section of the protocol above.--Bio-protocol team