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Fig. S1. Venus preservation assay.
Recombinant Venus proteins were incubated with neutralized chloral hydrate-based
were measured after 1-day incubation. Means ± standard errors are shown (n = 3).
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clearing solution, Scale-like solution, and ClearSee.v2. The fluorescent signal intensities
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Fig. S2. Optical clearing of Arabidopsis leaf using ClearSee.
solution (C) and chloral hydrate-based solution (D) for 4 days. Optical xz (left) and xy
(right) maximum-intensity projections were generated from 100 z-stack images with
1.0-µm intervals by CLSM with 488 nm excitation. The color bar indicates the depth
from the leaf surface. Scale bars, 100 µm.
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UBQ10pro::H2B-mClover leaves were treated with PBS (A), ClearSee (B), Scale-like
Fig. S3. Comparison of imaging penetration for CLSM and 2PEM in ClearSee-treated
Arabidopsis root tips.
RPS5Apro::tdTomato-LTI6b root fixed with PFA (Fixed) and treated with ClearSee for
4 days. Optical xy and xz sections were generated from 150 z-stack images with 1.0-µm
intervals by CLSM with 561 nm excitation (Confocal) and 2PEM with 950 nm
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excitation (Two-photon). Lower panels represent the cross-section at the position
indicated by the respective color lines (1, transition zone; 2, meristematic zone; 3,
columella). The top of the xz section images is facing the objective lens. Scale bars, 30
Fig. S4. Comparison of signal-noise ratio for CLSM and 2PEM in ClearSee-treated
Arabidopsis root tips.
The
fluorescence
intensities
of
RPS5Apro::tdTomato-LTI6b
at
the
cross-sectional lines (2, meristematic zone) shown in Fig. S3 were analyzed.
yellow
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µm.
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Fig. S5. Application of ClearSee for imaging of cellular components.
35Spro::mt-YFP (mt-yk) and 35Spro::GFP-mTalin (GFP-mTalin) leaves fixed with
PFA (Fixed) and treated with ClearSee (ClearSee) for 4 days. Mitochondria and the
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actin cytoskeleton were observed by 2PEM with 950 nm excitation. Scale bar, 100 µm.
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Fig. S6 Application of ClearSee for staining with chemical dyes.
The nuclei were stained with Hoechst 33342 in ClearSee-treated leaves. Optical xy (left)
and xz (right) maximum-intensity projections were generated from 144 z-stack images
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with 1.0-µm intervals by 2PEM with 800 nm excitation. Scale bar, 100 µm.
Fig. S7. Whole pistil imaging without ClearSee treatment.
(A) UBQ10pro::H2B-mClover pistil fixed with PFA. Optical xy and xz sections were
generated from 401 z-stack images with 1.0-µm intervals by 2PEM with 950 nm
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excitation.
(B)
Pistil
pollinated
with
LAT52pro::mTFP1,
LAT52pro::sGFP,
LAT52pro::Venus, and LAT52pro::mApple pollen fixed with PFA. Maximum intensity
projections for xy view and xz sections were generated from 141 z-stack images with
3.0-µm intervals by 2PEM with 990 nm excitation. Each image on the left in (A) and
the lower images in (B) represents the xz cross-sections at the position indicated by the
color lines shown as xz sections (1, stigma; 2, style; 3, ovary). The top of the xz section
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images is facing the objective lens. Scale bars, 100 µm.
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Fig. S8. Whole seedling imaging without ClearSee treatment.
SUC2pro::RCI2A-mCitrine seedling fixed with PFA. Maximum intensity projection for
nm excitation. Scale bar, 500 µm.
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xy view was generated from 85 z-stack images with 10-µm intervals by 2PEM with 950
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Table S1. List of clearing chemicals used for screening.
Click here to Download Table S1
Table S2. List of second screening combinations.
UBQ10pro::H2B-mClover leaves treated with ClearSee for 3 days. The fluorescence
intensity of H2B-mClover in the nucleus was measured with ImageJ. − = no
fluorescence, + = weak and +++ = highest fluorescent intensity (mClover fluorescence).
Observed autofluorescence intensity compared with PBS incubation: - = identical, + =
reduced, and ++ = weakest (Autofluorescence). Observed transparency: - = identical
with PBS incubation, + = weak and +++ = highest transparency (Transparency).
Table S3. List of third screening combinations.
For definitions see Table S2.
Click here to Download Table S3
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Click here to Download Table S2
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Movie 1.
A series of xz images of a root expressing DR5rev::3xVenus-N7 (green);
RPS5Apro::H2B-tdTomato (magenta) before fixation (Live) and after ClearSee
treatment for 4 days (ClearSee). Optical xz stack was generated from 150 z-stack images
with 1.0-µm intervals by CLSM with 488 nm and 561 nm excitations (1P) and 2PEM
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with 950 nm excitation (2P). View starts from the root tip. Scale bar, 30 µm.
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Movie 2.
SCRpro::GFP-SCR (latter) after fixation (Fixed) and after ClearSee treatment for 4 days
(ClearSee). Optical xy stack was generated from 101 (SCM) and 120 (SCR) z-stack
images with 1.0-µm intervals CLSM with 488 nm excitation (1P) and 2PEM with 950
nm excitation (2P). Scale bar, 50 µm.
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A series of xy images of a root expressing SCMpro::SCM-mGFP5 (former) and
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Movie 3.
Reconstituted 3D image of the leaf margin expressing DR5rev::3xVenus-N7 (green);
RPS5Apro::H2B-tdTomato (magenta) after 10-days ClearSee treatment. The 3D image
was reconstructed from 76 z-stack images with 1.0-µm intervals by 2PEM with 950 nm
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excitation. View starts from the adaxial side.
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Movie 4.
A series of xy images of a leaf margin expressing DR5rev::3xVenus-N7 (green);
RPS5Apro::H2B-tdTomato (magenta) without fixation. Optical xy stack was generated
from 100 z-stack images with 1.0-µm intervals by 2PEM with 950 nm excitation. Scale
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bar, 100 µm.
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Movie 5.
A series of xz images of pistil expressing UBQ10pro::H2B-mClover after 6-days
ClearSee treatment. Optical xz stack was generated from 410 z-stack images with
1.0-µm intervals by 2PEM with 950 nm excitation. View starts from the stigmatic
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papilla. Scale bar, 100 µm.
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Movie 6.
A series of xy images of a seedling expressing SUC2pro::RCI2A-mCitrine (green) after
7-days ClearSee treatment. Optical xy stack was generated from 283 z-stack images
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with 1.0-µm intervals by CLSM with 488 nm excitation. Scale bar, 100 µm.
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Movie 7.
Reconstituted 3D image of stem with cauline leaf expressing IPT3pro::GFP-GUS after
4-weeks ClearSee treatment. Images were acquired in sequential bandwidths of 8-nm
spanning the wavelength range of 460–648 nm to generate a lambda stack containing 19
images. The 3D image was reconstructed from 168 z-stack images with 1.0-µm
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intervals by 2PEM with 950 nm excitation. View starts from the adaxial side.
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Supplementary materials and methods
Microscopy settings
For screening, we used an upright microscope (BX-53; Olympus, Tokyo, Japan)
equipped with a charge-coupled device camera (DP73; Olympus). We used three filter
sets for long-pass (U-FBW; Olympus), band-pass (U-FBNA; Olympus) for mClover,
and long-pass for autofluorescence (U-FGW; Olympus) and the images were acquired
using a 10× objective lens (UPlanFL N, NA = 0.30, WD = 10 mm; Olympus). The
images were acquired and analyzed using the cellSens software (Olympus).
For deep imaging, we used a laser scanning inverted microscope (A1R MP;
Nikon, Tokyo, Japan) equipped with a Ti:sapphire femtosecond pulse laser (Mai Tai
DeepSee; Spectra-Physics, Mountain View, CA, USA). Z-stack images were acquired
using a 25× water-immersion objective lens (CFI Apo LWD 25× WI, NA = 1.10, WD =
2.00 mm; Nikon). For confocal imaging, 488-nm argon and 561-nm diode lasers were
used for excitation. Fluorescence signals were detected by a photomultiplier tube
detector. We used a dichroic mirror, DM560, and two band-pass filters, 525/50 nm for
Venus, mCitrine, or mClover, and 595/50 nm for tdTomato. A Ti:sapphire laser tuned
external non-descanned GaAsP PMT detectors. We used two dichroic mirrors, DM495
and DM560, and three band-pass filters, namely 479/40 nm for mTFP1, 534/30 nm for
GFP, Venus, mCitrine, or mClover, and 578/105 nm for tdTomato. Images were
processed with NIS-Elements AR software 4.10 (Nikon) to create maximum-intensity
projection images and to add color.
For moss imaging, we used a laser scanning inverted microscope
(LSM780-DUO-NLO; Zeiss, Göttingen, Germany) equipped with a diode-pumped
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to 950 nm was used for excitation of the FPs. Fluorescence signals were detected by the
Development 142: doi:10.1242/dev.127613: Supplementary information
solid-state 561 nm laser. Z-stack images were acquired using a 20× objective lens
(Plan-Apochromat, NA = 0.8; Zeiss). Each image was the average of two lines. Images
were acquired in the ranges 570–658 nm for H2B-mRFP and 672–701 nm for
autofluorescence. The images were acquired and analyzed using the ZEN 2010 software
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(Zeiss).