Plant Accessible Tissue Clearing Solvent System (PATCSOS) for 3-D Imaging of Whole Plants

Plant Accessible Tissue Clearing Solvent

System (PATCSOS) for 3-D Imaging of

Whole Plants

Hantao Zhang

, Lei Zhu

1,2*

, Hu Zhao

, Zhen Li

1,2*

College of Biological Sciences, China Agricultural University, Beijing 100193,

China

State Key Laboratory of Plant Environment Resilience, College of Biological

Sciences, China Agricultural University, Beijing 100193, China

Chinese Institute for Brain Research, Beijing, Beijing 102206, China

Correspondence: [email protected] (Z.L.); zhaohu@cibr.ac.cn (H.Z.);

[email protected] (L.Z.)

Abstract: Tissue clearing is a technique to make the inner structure of opaque tissue

visible to achieve 3-dimensional (3-D) tissue imaging by unifying the refractive

indexes of most of the cell components. Tissue clearing is widely used in animal

tissue imaging, where whole body 3-D imaging has been realized. However, it has not

been widely used in plant research. Most plant tissue clearing protocols have their

disadvantages, including low efficiency, not being fluorescence-friendly and poor

transparency on tissues with a high degree of lignification. In this work, we developed

a new plant tissue clearing method for whole plant imaging, named Plant Accessible

Tissue Clearing Solvent System (PATCSOS), which was based on the Polyethylene

Glycol-associated Solvent System (PEGASOS). The PATCSOS method realized

extensive transparency of plant tissues, including the flower, leaf, stem, root, and seed

of Arabidopsis thaliana, with high efficiency. The PATCSOS method consists of four

main steps: fixation, decolorization/delipidation, dehydration, and clearing.

Subsequently a rapid and efficient clearing of mature plant tissue can be achieved.

With PATCSOS, we can image Arabidopsis seedling in their entirety in 3-D using

endogenous cellulose autofluorescence. What’s more, the PATCSOS method is

compatible with fluorescence protein imaging and GUS staining, which greatly

expands the applicability of this method. We also imaged intact Nicotiana

benthamiana leaf and Zea mays embryos. Our results showed that the PATCSOS

clearing method is an excellent tool to study plant development and cell biology.

Keywords: tissue clearing; Arabidopsis thaliana; PATCSOS; 3-D imaging; confocal

microscopy

1 Introduction

To better investigate plant growth and development, it is important to obtain 3-D

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images of plant tissues and observe the inner structure of the plants. Different cell

components have different refractive indices (RI), for example, water has a RI of 1.33,

lipids have a RI of above 1.45, proteins have a RI of above 1.44, plant cell walls have

a RI of 1.42, and oxygen produced by green plants has a RI of about 1.00[1-5]. As a

result of these differences, the plant tissues appear to us to be opaque.

To address the issue of refractive index differences, the tissue transparency

technique was adopted a method of making the entire tissue transparent by unifying

the RI of most cellular components. Existing tissue clearing methods can be divided

into two categories: organic solvents and aqueous solution system[5, 6]. For example,

Visikol and Benzyl benzoate/benzyl alcohol (BABB) belong to the organic solvent

system [5, 7, 8]. In the BABB system, organic solvents, including benzyl alcohol and

benzyl benzoate, were used. iTOMEI, ClearSee and its derivative clearing methods,

and Pea-CLARITY used aqueous solutions[9]. These methods have many

disadvantages, such as poor transparency, long processing times, not being

fluorescence-friendly, and, most importantly, not being optimized for clearing plant

tissues.

In 2018, Hu Zhao and his team developed an effective tissue clearing method for

mouse whole body imaging called PEGASOS. PEGASOS can clear a wide range of

mouse tissues and PEGASOS system can protect the fluorescence signal from being

quenched [4]. Based on PEGASOS, we developed a plant accessible tissue clearing

solvent system (PATCSOS) and achieved considerable transparent effect for the whole

plant of Arabidopsis, which can warrant the construction of 3-D images of plant

tissues using autofluorescence from cellulose. We further confirmed that the modified

PEGASOS method is compatible with imaging with fluorescence protein and GUS

staining in plants.

2 Materials and Methods

2.1 Plant materials and growth condition

Arabidopsis thaliana (A. thaliana) accession Columbia-0 (Col-0) and transgenic

line ANTPro::GFP, DR5::GUS [10] and HB29Pro::GUS were constructed (see

below)The Arabidopsis seeds were surface-sterilized for 15 min in 0.5% sodium

hypochlorite and sown on 1/2 Murashige & Skoog medium containing 0.9% plant TC

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agar (PhytoTechnology Laboratories, Shawnee Mission, KS, USA). The Arabidopsis

grew at 22°C under a 16 h: 8 h, light: dark photoperiod.

2.2 Molecular cloning and transformation

A 2-kb region of the HB29 (AT1G69600) promoter and 2-kb region of the ANT

(AT4G37750) promoter were amplified. HB29 promoter was inserted into

pCAMBIA1391. ANT promoter, GFP gene and NOS terminator were inserted into

pCAMBIA1300 vectors to prepare HB29 Pro::GUS and ANT Pro::GFP constructs.

HB29 Pro::GUS and ANT Pro::GFP constructs were generated and transformed into

Col-0 plants to produce the marker lines. The plant transformation vector

pCAMBIA1300 and pCAMBIA1391 was used to generate transformed plants. Plants

were transformed using Agrobacterium tumefaciens strain GV3101 using the floral

dip method. [11]. The sequences of primers were listed in Table S1.

2.3 Preparation of PATCSOS solutions

The clearing solutions was adopted from PEGASOS with some modifications[4].

Fixation solution. Fixation solution was prepared by mixing 4% (w/v)

Paraformaldehyde (PFA, Sigma-Aldrich 158127) with 0.01 M PBS (pH 7.4, Solarbio

P1003).

Gradient tB solution.30%, 50% and 70% Tert-Butanol (tB, Aladdin T119717) in

water v/v and 3% w/v Quadrol (Sigma-Aldrich 122262) was added afterwards.

tB-PEG dehydration solution. 70% v/v Tert-Butanol (Aladdin T119717), 27% v/v

PEG methacrylate Mn 500 (PEGMMA500, Sigma-Aldrich 447943) and 3% w/v

Quadrol (Sigma-Aldrich 122262).

BB-PEG clearing medium. 75% v/v benzyl benzoate (BB, Aladdin B400547), 25%

v/v PEGMMA500 (Sigma-Aldrich 447943), and 3% w/v Quadrol (Sigma-Aldrich

122262).

2.4 Passive immersion procedure

Seedlings. Arabidopsis seedlings were immersed in 1 mL fixation solution and

fixed overnight at room temperature. The seedlings were washed with 1 mL of ddH

at 37℃ under gentle shaking for 20 minutes and repeated three times. Then they were

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immersed in a 30% tB solution for one day at 37℃ under gentle shaking to remove

the chlorophyll. Afterwards, the 30% tB solution was replaced with 50% tB for one

day and then with 70% tB for another day, both at 37℃ under gentle shaking. The

tissues were placed in a dehydration solution overnight at 37℃ under gentle shaking.

Finally, the dehydrated tissues were immersed in clearing medium for at least 24 h at

37℃ under gentle shaking.

Mature tissues. For mature tissues, the procedures were modified to fit the large

diameter and cellulose rich nature of the tissue. For bolting Arabidopsis, the

inflorescence stem was immersed in 4% paraformaldehyde (PFA) overnight at room

temperature, the tissues were washed three times with ddH

O, and then immersed in

30% tB for two days, and the 30% tB solution was replaced twice a day to better

remove chlorophyll. The stems were immersed in 50% tB for one day, 70% tB for one

day, and a dehydration solution for one day. And at last, they were immersed in the

clearing medium for at least one day. All above, the steps were performed on a

37℃shaker.

2.5 GUS Staining

Seven-day-old HB29pro::GUS seedlings were used to test compatibility between

PATCSOS and GUS staining. Seedlings were immersed in GUS assay solution (Table

S2) at 37℃ in the dark for 4 hours using a widely adapted protocol [12].

In the decolorizing step, the seedlings were immersed in 30% tB for 10 minutes,

50% tB for 10 minutes and 70% tB for 10 minutes to adapt to the PATCSOS system,

while in traditional methods, ethanol and acetate solution was used.

2.6 Microscopy and image analysis

GUS imaging was acquired on a Leica AF 6000 stereoscopic microscopy.

3-D fluorescence imaging of plant tissues was acquired on a Leica STELLARIS 5

confocal microscopy (Laser lines: 488, 561, 638; Leica HyD photon detector).

Raw images were collected in lossless 8-bit TIFF format. Single GFP

fluorescence images were processed with Fiji (NIH). 3-D reconstructed images were

generated using Imaris 9.0 (Bitplane). Stack images were generated using the “volume

rendering” function. 3-D image clipping was realized using the “Clipping Plane”

function. Background blocking was realized using the “Oblique Slicer” and “Ortho

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Slicer” function. 3-D pictures were generated using the “Snapshot” function. Movies

were made using the “Animation” function.

3 Results

3.1 Optimization of the PATCSOS procedure and evaluation of

clearing efficiency on plant tissues

There are five to six steps in the PEGASOS protocol, i.e.: fixation,

(decalcification), decolorization, delipidation, dehydration, and clearing [4]. The

decalcification step was omitted due to the absence of bone in the plant tissue. The

decolorization step can also be omitted as there is no heme in the plant tissue and the

gradient tB solution also has the effect of removing chlorophyll, therefore the steps of

delipidation and decolorization can be combined to reduce the complexity of the

method. The optimized PATGSOS protocol for plant tissue clearing included the

following steps: fixation, decolorization/delipidation, dehydration, and clearing.

Mature Arabidopsis inflorescence stems (Fig. 1b, 1e) became completely

transparent after 7 days in the clearing solution, the clearing duration is even shorter

for seedlings (Fig. 1a, 1d). After clearing, the inner vascular bundles can be seen

clearly in the inflorescence stem, and seeds in silique are also visible. This shows that

the pericarp has been completely cleared. Thinner plant tissues, such as leaves and

flowers, got the best transparent effect; they were completely invisible in the clearing

solution after processing (Fig. 1e).

We also cleared Nicotiana tabacum leaf and Zea mays embryo after germination.

The chlorophyll in tobacco leaf was removed effectively, and the whole leaf was

totally transparent after clearing (Fig. 1c, 1f). The embryo and endosperm of corn

seed was removed after clearing to get a better view of the corn embryo (Fig. 1g,1 h).

And after magnifying the embryo with a stereoscopic microscope, we can see the

developing plumule inside the coleoptile (Fig. 1i, 1j).

3.2 3-D imaging with autofluorescence

Autofluorescence is a normal phenomenon in plant tissues. Many cell

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components such as chlorophyll and cellulose can emit autofluorescence.

Autofluorescence is generally regarded as interference signal in fluoresce imaging and

various methods have been developed to remove it [13, 14]. But it serves as a natural

marker to show the cell profile and plant tissue structure. After clearing, we can

capture autofluorescence signal from cellulose clearly, with which a 3-D image of

plant tissue was obtained (Fig. 2).

We imaged flower of Arabidopsis with autofluorescence using 10×objective in

two channels (excitation: 488 nm and 561 nm), the step size was set at 1 m. Two

single slices at different Z-axis positions showing pollens in the anther sac and ovules

in the ovary, respectively (Fig. 2a, 2b). Clear observation of such tissues indicated

applicability of the PATCSOS for clearing flowers tissues. Reconstructed 3-D images

of flowers (Fig. 2e, 2f) can help us track the direction of the vascular system from

pedicel to stamens and pistil of the flower. So, the PATCSOS system can realize high

resolution 3-D imaging of fine structures of plants, regardless of inner or outer region

without dissection. Whole tissue clearing and 3-D imaging is more intuitive and can

maintain the relative position of each part compared with traditional dissection

methods [15].

More tender and more mature silique tissues were also imaged. Fig. 2c is one of

the slices of the tender silique captured with 40×objective stimulated by 488 nm

laser with a step size of 1m. Because of limited working distance of objective, we

only imaged half of a silique. The slicer tool in Imaris was used to reveal inner

structure of the silique, and the development condition of the embryo can be clearly

observed through the reconstructed image (Fig. 2g). Seeds in different developmental

stage and their relative positions in the silique can be clearly visualized (Fig. 2g1-g4).

A 20× objective with long working distance was used to image another more

mature silique with a small piece of stipe (Fig. 2i). Regretfully, because of the

flavonoid of the testa, the inner structure of some seeds was not clearly visible (Fig.

2i). Images and movie of silique in 3-D view showed continuous change from the

outer silique to the inner, and we can see the arrangement of seeds in a silique (Fig 2k,

Movie S1).

Part of inflorescence stem was imaged with a 10×objective stimulated by 488

nm laser, the step size was set as 1 m (Fig. 2d, 2h, 2j, 2l). The structure of vascular

bundles was clearly countable from the rendered 3-D image (Fig. 2l). The diameter of

the inflorescence stem was about 1 mm, the tissue needs to be highly transparent to be

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imaged at such high resolution and quality. This result showed that the PATCSOS

protocol has good clearing effect on Arabidopsis tissues imaging.

3.3 GFP fluorescence imaging

Fluorescence protein (FP) labelling is widely used to investigate tissue

development and protein subcellular localization in cell biology [5]. In whole tissue

clearing analysis, the ultimate goal was to image the inner structure of tissues using

fluorescence； thus, the protection of fluorescent proteins during clearing is of critical

importance.

The passive immerse process employed by PATCSOS can readily protect the

fluorescent proteins by using gradient tB to replace ethanol in traditional plant

material processing methods and adding Quadrol to keep a basic environment to

maintain activities of the FPs. However, GFP imaging of Arabidopsis line ANT

Pro::GFP presented the dilemma of discriminating fluorescence signal between

autofluorescence and GFP. Fluorescence from FPs can only be stimulated by

excitation light with specific wavelength. In plants, chlorophyll and lignin stand out as

the key auto fluorescent substances, yet a variety of additional compounds exhibit

autofluorescence when subjected to ultraviolet or visible light excitation,

encompassing elements from the cytoplasm and the structural cell walls [16]. While

for autofluorescence, a broad wavelength range can be used for excitation. Thus,

during image acquisition, we collected data from two channels, the first channel used

488 nm laser for excitation and signals from both GFPs and autofluorescence were

acquired, the other channel used 561 nm laser for excitation and signal only from

autofluorescence was acquired. Images from these two channels were subtracted to

obtain images only from the fluorescence proteins [17].

Fluorescence images from one section of Arabidopsis ANTPro::GFP stipe was

imaged with excitation laser at 488 and 561 nm, the images showed distinct

distribution of fluorescence signals from GFP and autofluorescence (Fig. 3a , 3b).

When a 488 nm laser was used, fluorescence from both GFP and autofluorescence can

be clearly visualized from cambium and epidermis region of the stipe (Fig. 3a). When

a 561 nm laser was used, fluorescence only from epidermis can be imaged (Fig. 3b).

Since ANT is a marker gene to label the cambium[18], the ANTPro::GFP construct

should emit fluorescence signals only from the cambium region, but fluorescence

image from the 488 nm channel showed ubiquitous distribution of signal due to

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interference from autofluorescence from cellulose (Fig. 3a). We further merged

images from the two channels (Fig. 3d), to distinguish autofluorescence from GFP

signal, the orange region represents autofluorescence signal from the 488 nm and 561

nm channels and the green region represents GFP signal from only the 488 nm

channel.

We further used “Subtract” function of the Fiji software remove autofluorescence

interference signal. The brightness of the two images was adjusted to the same level,

and the “Subtract” function was used to performance subtraction between Fig. 3a and

3b, which resulted in a clean GFP fluorescence image of Arabidopsis cambium (Fig.

3e).

Through 3-D imaging reconstruction and background subtraction, a constant ANT

express pattern in the whole stipe was observed (Fig. 3c, 3f), where, the yellow part

represents autofluorescence and the green part represents GFP signal, and the

cambium pattern in the stipe can be clearly visualized.

3.4 Whole seedling 3-D imaging

3-D imaging of whole Arabidopsis seedling was also realized with PATCSOS.

The immature development status of cell wall makes the seedling tissue tenderer than

mature inflorescence stem, turgescences from vacuole provides support for the cell

instead of cellulose in cell walls. Thus, maintaining cell shape during the clearing

process is the critical step for clearing seedlings. We modified the PATCSOS protocol

by shortening the decolorization/delipidation time and controlling dehydration time

strictly to fit the tender nature of seedlings and achieved excellent clearing effect

while maintaining cell shape. Hence, the shoot apical meristem (SAM) and trichome

can stay in good status after clearing (Fig. 4i, 4j). Eight-day Col-0 seedlings were

cleared with the modified PATCSOS method and loaded onto a glass slide with a

central groove. The whole seedling was imaged with a 40×oil objective using 488

nm excitation laser with a step size of 1 m. The image of the seedling vascular

bundle showed that the annular vessel and spiral vessel are the major vessel elements

in the vascular bundle (Fig. 4a). An image of part of the shoot apical meristem (SAM)

clearly showed cell configuration details of the bud primordium (Fig. 4b). When the

laser was focused at the root platform, the root hair could be clearly visualized (Fig.

4c, 4c1), when the laser was focused at the SAM platform, bud primordium could be

clearly visualized (Fig. 4d, 4d1), both images were processed by the brightness adjust

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function of the Fiji software. 3-D imaging of whole seedling showed clear details of

all the root hairs, which also proved that small details can be well preserved by

processing with PATCSOS (Fig. 4e, 4f, 4g). A movie was made to show more details

of the whole seedling (Movie S2). A slice image in the z-direction of the diarch stele

in Arabidopsis root showed the xylem and phloem arrangement (Fig. 4h). Details of

the surfaces of cotyledon and SAM can be clearly visualized, and the structure of the

trichomes was also well-preserved (Fig. 4j). The modified PATCSOS method realized

whole tissue 3-D imaging of Arabidopsis seedling without the need of dissection,

which is perfectly suitable for 3-D imaging of small and tender tissues such as

seedlings, as such tissues are generally very soft and small in diameter which render

them difficult to dissect/slicing.

3.5 GUS staining after clearing

GUS staining is a widely used technology to reveal gene expression position and

expression level. But in some non-cleared tissues, GUS staining may be difficult to

observe due to the opaque nature of plant tissues. Thus, the combination of GUS

staining and tissue clearing is a perfect way to realize GUS staining from deep-buried,

opaque tissue regions. Here, we tested the compatibility of the PATCSOS system with

GUS staining. Eight-day seedlings were first stained with conventional GUS staining

solution [19]. To adapt to the PATCSOS system, the tissues were decolorized at 30%,

50%, and 70% tB for 10 minutes, respectively, instead of ethanol in the conventional

GUS staining protocol. The decolorized seedling can be cleared in less than 12 h by

immersing in a standard PATCSOS clearing solution.

HB29 is a gene we were concerned about at the earlier time. And DR5 is a famous

auxin respond element.[20] So, we chose these two genes to test our protocol.

HB29Pro::GUS seedling was first GUS stained and then processed with

PATCSOS-GUS clearing, the difference in contrast between tissues is more clearly

(Fig. 5a, 5c). DR5::GUS seedling were also treated with GUS staining and then with

PATCSOS-GUS clearing (Fig. 5b, 5d), some edge areas with unclear coloring in

traditional GUS staining were now clearly visible. Thus, the PATCSOS-GUS clearing

system can maintain GUS staining after clearing and the higher contrast between GUS

staining and cleared tissue makes high resolution 3-D imaging available.

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4 Conclusions and Discussion

In this work, we modified the popular mouse tissue clearing method PEGASOS

to suit application in plant science research and developed the PATCSOS protocol.

The PATCSOS procedure is FPs friendly and can maintain tissue structure of tender

Arabidopsis seedlings. We also confirmed that PATCSOS is compatible with GUS

staining using the modified PATCSOS-GUS protocol.

With the developed method, 3-D images of different plant organs were acquired.

A new concept for autofluorescence imaging was proposed, which uses

autofluorescence from cell wall components for 3-D imaging, and the

autofluorescence signal has generally been considered as noise signal. With

autofluorescence, 3-D image from an intact seedling was obtained. By acquiring

fluoresce signals from different excitation channels, fluorescence signals from

autofluorescence and FPs can be discriminated to reveal “true” signal from the FPs, in

this way we imaged the cambium pattern in Arabidopsis stipe.

The PATCSOS protocol still has some disadvantages, tB was used for

decolorization/delipidation, but, compared with ethanol, the chlorophyll removing

efficiency of tB is much lower, but ethanol will quench the FPs fluorescence. So, it is

necessary to find a fluorescence friendly and effective chlorophyll removing reagent.

And for seeds, which is regarded as the most difficult organ to be cleared, our current

protocol still has difficulties to fully clear seeds. Light-sheet microscopy is an ideal

technology to image whole mature plant, but we found that the light beam was

scattered badly by the vascular tissue. Thus, a better method to control light scattering

by the vascular bundle is needed to realize 3-D imaging with light-sheet.

5 Supplementary Materials

The following are available in supplementary document.

Table S1: Primers used in this study;

Table S2 GUS assay solution (50 ml);

Table S3 X-Gluc mother liquor (250 μL).

Movie S1: 3-D imaging of Silique;

Movie S2: Whole seedling 3-D imaging presentation.

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6 Author Contributions

Zhang HT. designed the project, performed the experiments and imaging, and

wrote the original draft; Li Z., Zhao H., Zhu L. reviewed and edited the article. All

authors have read and agreed to the published version of the manuscript.

7 Acknowledgments

We thank Beijing Students' Platform for innovation and entrepreneurship training

program for providing apart of financial support. We also thank Yutao Wang, Huan

Zhao, Hongjie Xing (China Agricultural University) for help in construction of

transgenic plant material, and Youqi Li, Yuling Wang, Manyu Chen, Jiayi Ding

(Chinese Institute for Brain Research, Beijing) for the imaging technology assistance.

8 Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Different plant materials treated by PATCSOS. Arabidopsis seedlings were visible in PFA (a)

without clearing, and after PATCSOS treatment, the seedlings became invisible (d). So as the mature

Arabidopsis plant, a part of the inflorescence stem(b) also can be cleared by PATCSOS (e). Nicotiana

tabacum leaf (c) and germinated Zea mays embryo (g) before (c, g) and after (f, h) processed with

PEGASOSFP. At the center of the embryo, the developing plumule can be seen (i, j). Bar, 1 cm (a, d, g,

h); bar, 5 cm (b, e); bar, 2 cm (c, f); bar, 1 mm (i, j).

The copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.20.590386doi: bioRxiv preprint

Figure 2, 3-D imaging of Arabidopsis tissues using autofluorescence: (a-b) two images of single light slices of

flowers with different focal planes; (e-f) 3-D reconstructed images of flowers; (c) single slice of silique; (g) 3-D

reconstructed image to highlight the embryo; g1-g4: magnification of the image framed by the white dotted line

in (g); (i) single slice of a mature silique; (k) 3-D reconstructed image of the mature silique in (i); (d, h) single

slices of inflorescence stem focused on different depth of focus; (j, l) different view of the inflorescence stem

3-D image, and vascular bundles are labeled by arrows in (l).

Most autofluorescence was excited using 488-nm

argon lasers, and the flower tissue imaging was also excited using 561-nm laser. Bar, 1 mm (a, b, c, d, h, i, k);

bar, 300 μm (e, f, g, l); bar, 100 μm (g1, g2, g3, g4); bar, 500 μm (j).

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Figure 3 Confocal imaging of the stipe of ANTPro::GFP transgenic Arabidopsis. The GFP

fluorescence (a) and the autofluorescence (b) imagines are captured. And merge (d), subtract (e) and

view them in 3-D vision (c, f). Two channels were used for imaging. Channel 1 used 488-nm laser as

exciting light, and emission light was collected from 499.8-579.3 nm. Channel 2 used 561-nm laser as

exciting light, and emission light was collected from 623.5-725.7 nm. Bar, 250 μm (a, b, d, e); bar, 200

μm (c, f).

The copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.20.590386doi: bioRxiv preprint

Figure 4 3-D imaging of Arabidopsis whole seedling: (a) vascular bundle in hypocotyl; (b) a prat of

SAM; (c, d) two images of single light slices of seedling focused on different depths; (c1) root hairs, a

magnification of the white dashed box in (c); (d1) bud primordium, a magnification of the white dashed

box in (d); (e-g) different views of whole seedling 3-D image; (h) Z direction view of the seedling root;

(i) an image of cotyledon; (j) an image of trichome. Bar, 1 μm (a, b); Bar, 1 mm (c, d, e, f, i); bar, 100

μm (c1, d1, j); bar, 500 μm (g); bar, 300 μm (h).

The copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.20.590386doi: bioRxiv preprint

Figure 5 Comparison between traditional GUS staining and PATCSOS-GUS staining: (a) HB29Pro::GUS

stained by the traditional method; (b) DR5::GUS stained by the traditional method; (c) HB29Pro::GUS

stained by the PATCSOS-GUS method; (d) DR5::GUS stained by the PACTSOS-GUS method. Bar, 1 mm.

The copyright holder for this preprintthis version posted April 22, 2024. ; https://doi.org/10.1101/2024.04.20.590386doi: bioRxiv preprint