基于HEMA-AAm共聚物的快速无溶剂组织透明化方法用于荧光成像

Yanran Wang; Siying Feng; Xiaoqi Zhou; Qiufeng Yao; Hui Ma; Kefeng Wu

doi:10.21769/BioProtoc.5497

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Peer-reviewed

Rapid and solvent-free, 2-hydroxyethyl methacrylate (HEMA)-acrylamide (AAm) copolymer-based optical clearing of tissue for fluorescent imaging

基于HEMA-AAm共聚物的快速无溶剂组织透明化方法用于荧光成像

KW Kefeng Wu email

发布: 2025年11月20日第15卷第22期 DOI: 10.21769/BioProtoc.5497 浏览次数: 58

评审: Olga KopachAnonymous reviewer(s)

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Apr 2025

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Abstract

The study of whole organs or tissues and their cellular components and structures has been historically limited by their natural opacity, which is caused by the optical heterogeneity of the tissue components that scatter light as it traverses through the tissue, making 3D tissue imaging highly challenging. In recent years, tissue clearing techniques have received widespread attention and undergone rapid development. We recently demonstrated the synthesis of a 2-hydroxyethyl methacrylate (HEMA)-acrylamide (AAm) copolymer. This was achieved using antipyrine (ATP) and 2,2′-thiodiethanol (TDE) as solvents. The resulting solution rapidly embedded tissue samples with a high degree of transparency and is compatible with multiple fluorescence labeling techniques. The method exhibits significant transparency effects across a range of organs, comprising the heart, liver, spleen, lung, kidney, brain (whole and sectioned), esophagus, and small intestine. It can enable volumetric imaging of tissue up to the scale of mouse organs, decrease the duration of the clearing, and preserve emission from fluorescent proteins and dyes. To facilitate the use of this powerful tool, we have provided here a detailed step-by-step protocol that should allow any laboratory to use tissue transparency technology to achieve transparency of tissues and organs.

Key features

• The method is primarily used for optical tissue clearing.

• The method employs a HEMA-AAm copolymer for optical tissue clearing.

• This method enables both 2D and 3D fluorescence imaging.

Keywords: Copolymer (共聚物)

Tissue embed (组织包埋)

Tissue clearing (组织透明化)

Optical transparency (光学透明性)

Fluorescence labeling (荧光标记)

Volumetric imaging (体积成像)

Background

There is a growing demand in the biological field for 3D volume imaging deep in biological tissues [1], but the optical heterogeneity of the tissue components results in opacity, which causes scattering of light as it penetrates the tissue [2] and makes 3D deep tissue imaging highly challenging [3]. A simple way to bypass tissue opacity is to section the thick tissue into several thin slices. Conventional tissue sectioning, however, is a laborious and time-intensive procedure and often causes tissue deformation during section preparation. Tissue-clearing techniques and high-resolution volumetric imaging by fluorescence microscopy [4,5] with appropriate fluorescent labeling can provide comprehensive cell information, such as cell type, shape, state, and distribution of cells of interest throughout an organ [6–10]. In particular, tissue-clearing techniques combined with fluorescence microscopy are a powerful tool to quantify rare cells such as stem cells, metastatic cells, or activated neurons in a whole organ for biological research and pathological diagnosis [11]. Hydrogel-based tissue clearing methods (e.g., CLARITY [12,13], PACT [14,15], SHIELD [16], SWITCH [17]) stand out for their stability and compatibility with bio-molecular staining. The intact biological tissues are replaced by a hydrogel polymerization framework, where the proteins, nucleic acids, and other macro-biomolecules are immobilized at their native physiological positions, while the lipid components remain unbound and are able to be eluted [18,19]. The decrease of the lipid barrier increases the penetrability of light and antibodies, allowing the direct stereo imaging of structural and molecular phenotyping of samples. In addition, the firm and stable hydrogel environment limits the diffusional contact between the indicator dye and reactive species, thereby improving shelf life and photochemical stability of the fluorescently labeled specimens, which is particularly important in high-resolution 3D imaging [20]. Enormous efforts have been made to fabricate hydrogels with enhanced mechanical properties by using hybrid hydrogels [21,22] or nano-composite hydrogels or by designing distinctive structures such as an interpenetrating network and dual-crosslinking network. By adjusting the hydrogel’s composition, the chemical environment of the tissue-hydrogel complex can be engineered to create a favorable condition for tissue clearing [23].

The 2-hydroxyethyl methacrylate-acrylamide (HEMA-Aam) method significantly outperformed most hydrogel-based clearing techniques, particularly in terms of clearing speed, tissue transparency, and size preservation. This approach effectively preserves tissue morphology and is compatible with fluorescence labeling, making it optimal for high-resolution imaging and long-term studies. The HEMA-AAm method achieved a high level of transparency in centimeter-scale mouse kidneys within four days. In comparison, the PACT method achieved similar transparency in approximately six days, while the TESOS method required several weeks. However, the TESOS method, despite offering relatively good transparency, exhibited high toxicity and caused severe fluorescence quenching. The PACT method displayed the poorest overall performance and lacked practical utility. During the process, we observed an increase in kidney tissue size from 100% (PBS) to 110% ± 2%, indicating negligible tissue expansion to maintain the size of the samples caused by the immersion and embedding processes. In contrast, the TESOS and PACT methods induced significant expansion or contraction of the samples. This transparent and hydrogel condition provides a friendly tissue and cellular environment to facilitate high-resolution 3D imaging, preservation, transfer, and sharing among laboratories to investigate the morphologies of interest in experimental and clinical conditions. Here, we offer a comprehensive and progressive process to facilitate the transparent handling of tissues and organs. We also describe how to handle tissue samples, including the preparation of immunofluorescence staining and the preparation of hydrogels.

Materials and reagents

Biological materials

1. 8–12-week-old wild-type C57BL/6 mice (Guangdong Laboratory Animal Monitoring Institute)

2. Enhanced green fluorescent protein (EGFP) transgenic mice (Jiangsu Huachuang Xinnuo Medical Technology Co, Ltd.)

Reagents

1. Antipyrine (ATP) (Shanghai Macklin Biochemical Co, Ltd, catalog number: 60-80-0)

2. Acrylamide (AAm) (Shanghai Macklin Biochemical Co, Ltd, catalog number: 79-06-1)

3. Poly(ethylene glycol) diacrylate (PEGDA) (Shanghai Macklin Biochemical Co, Ltd, catalog number: 25736-86-1)

4. 2,2'-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044) (Shanghai Macklin Biochemical Co, Ltd, catalog number: 27776-21-2)

5. Thiodiglycol (TDE) (Kryptonite Ltd, catalog number: CD101467)

6. 2-Hydroxyethyl methacrylate (2-HEMA) (Aladdin, catalog number: 868-77-9)

7. 4% paraformaldehyde (PFA) (Leagene, catalog number: DF0135)

8. 0.02% sodium azide (Aladdin, catalog number: 26628-22-8)

9. 2% Triton X-100 (Aladdin, catalog number: 9036-19-5)

10. CD31 antibody [Signalway Antibody (SAB), RRID: AB_852501]

11. Perilipin A Rabbit mAb [Signalway Antibody (SAB), RRID: AB_2863342]

12. Goat anti-rabbit lgG secondary antibody AF488 conjugated (Jackson ImmunoResearch, RRID: AB_3246434)

13. Lectin (Vector Labs, RRID: AB_2314736)

14. DAPI (Sigma-Aldrich, RRID: AB_2869624)

15. PBS (Servicebio, catalog number: G0002)

16. Isoflurane (Nanjing Aibei Biotechnology Co, Ltd, catalog number: 2409A)

17. Primary antibody dilution buffer, QuickBlock^TM immunostaining primary antibody dilution buffer (Beyotime, catalog number: P0262)

18. Secondary antibody dilution buffer, QuickBlock^TM immunostaining secondary antibody dilution buffer (Beyotime, catalog number: P0265)

19. Blocking buffer, QuickBlock^TM immunostaining blocking buffer (Beyotime, catalog number: P0260)

Solutions

1. Tissue clearing solution (see Recipes)

2. Monomer solution (see Recipes)

3. Prepolymer solution (see Recipes)

4. 0.01 M PBS (see Recipes)

Recipes

1. Tissue clearing solution

Reagent	Final concentration	Quantity or Volume
ATP	348 g/L	1.8823 g
TDE	450 g/L	2.062 mL
Distilled water	216 g/L	1.0815 mL
Total	998 g/L	~5.4 mL

2. Monomer solution

Reagent	Final concentration	Quantity or volume
AAm	-	0.3 g
2-HEMA	0.1 mL	1.82 mL
PEGDA	0.1 mL	0.155 mL
Total	-	-

Critical: 2-HEMA and PEGDA solutions are first mixed together, and then 1 mL of the mixture is taken and added to the subsequent prepolymer solution.

3. Prepolymer solution

Reagent	Final concentration	Quantity or volume
Monomer solution	404.9 g/L	0.3 g + 0.1 mL
Tissue clearing solution	1,062 g/L	0.9 mL
Total	1,466.9 g/L	~1.0 mL

Critical: Following preparation of the prepolymer solution, a 1 mL aliquot is mixed with 15 μL of VA-044 to induce gel formation through polymerization.

4. 0.01 M PBS

Reagent	Final concentration	Quantity or volume
PBS powder	0.01 M	One sachet (commercially provided)
Distilled water	-	2 L
Total	-	2 L

Note: The PBS solution was prepared by dissolving commercially obtained PBS powder in 2 L of deionized water.

Laboratory supplies

1. 1,000 μL pipette tips (BioSharp, catalog number: 24824031E)

2. 1,000 μL pipette (Thermo Fisher, catalog number: MZ11332)

3. 100 μL pipette tips (BioSharp, catalog number: 21223639E)

4. 100 μL pipette (Thermo Fisher, catalog number: QZ55048)

5. 20 μL pipette (Thermo Fisher, catalog number: TO19723)

6. 24 × 24 mm standard-grade microscope coverslips (Citotest, catalog number: 10212424C)

7. 7 × 7 × 5 mm embedding cassette base mold (Citotest, catalog number: 80203-0006)

8. 15 mL centrifuge tube (BioSharp, catalog number: 30324124E)

9. 50 mL centrifuge tube (BioSharp, catalog number: 35224125E)

10. 5.0 mL microcentrifuge tube (BioSharp, catalog number: 23029335A)

11. 2.0 mL microcentrifuge tube (BioSharp, catalog number: 241211)

12. Adhesive (Pattex, catalog number: 24032809)

13. Pasteur pipette (Kangpeite, catalog number: 20240136)

14. 30 mL sterile syringe (Hongda, catalog number: 20193141735)

15. 1 mL sterile syringe (Hongda, catalog number: 20193141735)

16. Disposable intravenous infusion needle (Hongda, catalog number: 20173144093)

17. Cryostat [Dakewe (Shenzhen) Medical Equipment Co., Ltd., model CT520]

Equipment

1. Light-sheet fluorescence microscope (Nuohai, model: LS18)

2. Digital ultrasonic cleaner (Kangshijie, model: PL-S40T)

3. Air-bath thermostatic shaker (Jinyi, model: THZ-82B, serial number: XMTD-702)

4. Confocal microscope (Olympus, model: IXplore spinSR)

5. Horizontal rotator (Taizhou Nuomi Medical Technology Co, Ltd, model: NMYC-100)

6. Automatic refractometer (Shanghai Yimai Instrument Technology Co, Ltd, model: IR140)

7. Cryostat stage (MEDITE Medical, Leica CM1950)

8. Confocal microscope (IXplore spinSR, Olympus, Japan)

Software and datasets

The data in the figure and legend of the manuscript are presented as mean ± standard deviation (x ± s). Statistical analysis was conducted using t-tests or one-way analysis of variance (ANOVA) as appropriate. Statistical analyses were performed using ImageJ, GraphPad Prism and origin, 3D reconstruction was performed using AIVIA (version 14, Manufacturer Information:Leica Microsystems (Shanghai) Trading Co., Ltd.). All data and analysis needed for the development and characterization of this protocol are available in the main text or Supplemental Information of Qin et al. [24].

Procedure

English

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稿件历史记录

提交日期: Jul 30, 2025

接收日期: Sep 19, 2025

在线发布日期: Oct 17, 2025

出版日期: Nov 20, 2025

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Wang, Y., Feng, S., Zhou, X., Yao, Q., Ma, H. and Wu, K. (2025). Rapid and solvent-free, 2-hydroxyethyl methacrylate (HEMA)-acrylamide (AAm) copolymer-based optical clearing of tissue for fluorescent imaging. Bio-protocol 15(22): e5497. DOI: 10.21769/BioProtoc.5497.