Optical Clearing and Confocal Imaging

We mounted samples with 10 μl of 1% agarose gel on a glass-based dish (Greiner Bio-One, 627871) for stable imaging. Then, the samples were immersed with the BABB solution (benzyl-alcohol and benzyl-benzoate, 1:2) or CUBIC2 solution for optical clearing (Hirashima and Adachi, 2015 (link); Susaki et al., 2014 (link); Yokomizo et al., 2012 (link)). Finally, we obtained 8-bit volumetric fluorescence images using the confocal laser-scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD. We used objective lens magnifications of ×20 [numerical aperture (NA)=0.75, working distance (WD)=680 μm, HC PL APO CS2, Leica], ×40 (NA=1.3, WD=240 μm, HC PL APO CS2, Leica) or ×60 (NA=1.4, WD=140 μm, HC PL APO CS2, Leica).

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Hirashima T, & Adachi T. (2019). Polarized cellular mechano-response system for maintaining radial size in developing epithelial tubes. Development (Cambridge, England), 146(23), dev181206.

Publication 2019

Leica TCS SP8 Leica HyD HC PL APO CS2

Agarose Benzyl alcohol Benzyl benzoate Cubic2 Dish Fluorescence Hybrid Lens

Corresponding Organization :

Other organizations : Kyoto University

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Variable analysis

independent variables

Type of optical clearing solution (BABB or CUBIC2)

dependent variables

Volumetric fluorescence images
Pixel intensity of fluorescence images

control variables

Sample preparation (10 μl of 1% agarose gel on glass-based dish)
Confocal laser-scanning platform (Leica TCS SP8 with hybrid detector Leica HyD)
Objective lens magnifications (×20, ×40, or ×60)

controls

No positive or negative controls were explicitly mentioned.

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