Inagaki, S., Nakagawa-Tamagawa, N., Huynh, N.Z. et al. Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo. Nature Methods (2026). https://doi.org/10.1038/s41592-026-03023-y
[bioRxiv preprint]
[Research Briefing (Nature Methods)] [EurekAlert!] [FocalPlane] [The Transmitter]
Raw image data: https://ssbd.riken.jp/repository/484/
The reagent (SeeDB-Live/ACSF) is commercially available from Nacalai Tesque. Please contact them if you want to try.
Step-by-step protocol (PDF in Google Drive)
Please contact us for any questions (imai.takeshi.457@m.kyushu-u.ac.jp).
For the repeated in vivo imaging with a plastic film window, see also Manita et al. (2022).
Please note that the refractive index of SeeDB-Live is 1.363, which is higher than water (1.33) but lower than silicone (1.41).
It is strongly recommended to use objective lenses with a correction collar (water or multi-immersion) to minimize spherical aberrations. We used XLPLN25XWMP (Evident/Olympus) and LUPLAPO25XS (Evident/Olympus).
It is also recommended to use the immersion of the same index (e.g., 15.6% TDE in H2O, RI 1.363). You cannot make the best use of SeeDB-Live with a water-immersion lens without correction collar, because of the spherical aberrations (RI 1.33 vs 1.363); the transparency will increase with SeeDB-Live, but the brightness and resolution will decrease.
We have been successful with some samples but not with others. Importantly, BSA cannot easily penetrate physical barrier (dura mater, tight junction, etc.). In addition, the high concentration of BSA may limit diffusion of nutrients, oxygen, and cytokines, which may affect normal growth of some types of organoids.
HeLa cell spheroids, intestinal organoids, primary culture of neurons, acute brain slices, mouse cerebral cortex in vivo (after durotomy)
Optic cup induction from ES cells: Growth factors/cytokines in the media may be critical for the long-term induction process. Acute functional assay is possible for ES/iPS-derived organoids.
Long-term culture of organoids: Growth was often affected possibly due to limited diffusion of nutrients and oxygen. We typically limited the SeeDB-Live treatment up to 4 hours/day.
Epithelial tissues: SeeDB-Live cannoe easily penetrate tight junction. Therefore, it is recommended to cut the epithelial organoids into pieces.
Myelin-rich brain regions (brainstem, nodose ganglion, etc.): Refractive index of lipid is much higher than SeeDB-Live.
Subcortical regions (hippocampus, striatum, hypothalamus, etc.): Passive diffusion of SeeDB-Live is typically limited to the cerebral cortex. We have not yet achieved 2P imaging of subcortical regions.
Skull should be better cleared with other invasive methods (such as SeeThrough and HOTS).
Coming soon!