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Visualize Embryogenesis and Cell Fate Using Fluorescent Probes with Aggregation-Induced Emission Fang Hu, Purnima Manghnani, * Kenry, Guangxue Feng, Wenbo Wu, Cathleen Teh, and Bin Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19391 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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ACS Applied Materials & Interfaces

Visualize Embryogenesis and Cell Fate Using Fluorescent

Probes

with

Aggregation-Induced

Emission

Fang Hu†#, Purnima Naresh Manghnani†#, Kenry†, Guangxue Feng†, Wenbo Wu†, Cathleen Teh§, and Bin Liu†* †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, 117585, Singapore §

Institute of Molecular and Cell Biology, Biopolis, Singapore 138673, Singapore

ACS Paragon Plus Environment

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ABSTRACT

Horseradish peroxidase (HRP) and fluorogen-dextran conjugate are tracers extensively used for injection-based lineage tracing. However, HRP is sensitive to proteolytic digestion while the highmolecular-weight dextran may have antigenicity. Small molecular tracers can overcome these problems, but they usually diffuse from labeled cells, causing inaccurate information. Herein, we developed a small-molecular-weight fluorogen with aggregation-induced emission (AIEgen) for embryonic cell tracing with strong signals against tracer dilution caused by cell division. Once injected into the ancestor cells, the AIEgen can be entrapped in the cells without leakage because of the two hydrophilic and neutral arms. Consequently, it can specifically trace the progenies of the treated ancestor cells. More importantly, the operating concentration of AIEgen can be much higher than those of fluorogens with aggregation-caused quenching (ACQ), which provides bright signals in daughter cells during embryonic cell tracing, thus overcoming the problem of fast signal degrading typically encountered with the use of traditional cell tracers.

Keywords: aggregation-induced emission • small molecule • bright signals • embryonic cell • zebrafish lineage tracing • cell entrapped

INTRODUCTION Lives mostly begin with the proliferation and differentiation of fertilized egg cells. Visualization of embryonic cell functions can greatly help for deep understanding of life origin and disease prevention. Lineage tracing is a technique used to identify the progeny of a single cell or group of cells in complex bio-systems.1 It can provide information about the number, location


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and differentiation status of the progenies of ancestor cells, which are directly related to life formation processes.2 Traditionally, lineage tracing has been widely used in developmental biology to unravel the development of complex organism from embryonic cells.3-6 Recently, this technique has also been increasingly applied to track stem cell dynamics,7 as well as those of cancer stem cells,8 heterogeneity of tumor cells,9 production of T cells in disease responses,10 etc. Nowadays, there are many strategies of lineage tracing, which include direct observation, vital dye labeling, intracellular injection of tracers, and transfection of genetic markers.2 Most of these strategies can only enable tracking of the lineage of cell groups which often label unrelated neighboring cells and pose the problem of yielding inaccurate information. Since external nano/micro-injection can accurately administrate target single-cell with tracers, it enables precise tracking of single-cell proliferation and differentiation.6, 11 Up to now, several types of tracers have been utilized for nano-/micro-injection-based lineage tracing of embryonic cells, which include genetic markers,12 enzymes, i.e. horseradish peroxidase (HRP)11 and fluorogens conjugated to high-molecular-weight dextran.13 Genetic markers that express fluorescent proteins do not spread to neighboring cells and exhibit high specificity to progenies of injected cells. However, the expression efficiency of injected plasmid is generally low, which may result in unsuccessful tracing.12 Luckily, enzymes and fluorogen-dextran conjugates do not have this problem. The progenies of the injection-treated ancestor cells can be labeled with exogenous HRP or fluorogen-dextran conjugate, realizing precise tracking of single embryonic cell proliferation and differentiation. In addition, they are sufficiently large to prevent diffusion to adjacent cells. As HRP is sensitive to proteolytic digestion,6 fluorogen-dextran conjugate is the most popularly used tracer for injection-based lineage tracing. However, it was reported that the higher the molecular weight of dextran, the more likely it could be antigenic.14


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Due to the molecular weight distribution of dextran, the preparation of fluorogen-dextran faces the challenge of batch to batch variation.15 Direct use of small-molecular-weight tracers may address these issues and also provide very uniform images, but they generally face diffusion problems. Therefore, biocompatible and small-molecular-weight fluorogen with no cell leakage is highly attractive for the development of lineage tracing technique. The operating concentration of conventional fluorogens for lineage tracing is low (within 5 μM) because of aggregation caused quenching (ACQ) effect. Consequently, the signal of conventional fluorogens fades quickly due to rapid cell division-induced dilution during lineage tracing. Aggregation-induced emission (AIE)16 is a phenomenon that is the opposite of ACQ effect. Fluorogens with aggregation-induced emission (AIEgens) are almost not emissive in molecularly dissolved state but emit strongly in aggregate state or at high concentrations. Therefore, AIEgen nanoparticles have been successfully used to provide fluorescent signals for long-term tracking with good photostability.17 In addition, molecular AIEgens can be used at high concentration and emit brightly upon interaction with specific biomolecules in bio-systems, as a result of the activation of AIE.17, 18 With all these outstanding features, AIEgens have been widely used for cell imaging19-21 and intracellular bioprocess monitoring.22-27 Motivated by the tracer demand in lineage tracing and the promising performance of AIEgens, herein we designed a small-molecular-weight AIEgen, namely (E)-4-(4-(2,2-bis(4-(2-(2-(2hydroxyethoxy)ethoxy)ethoxy)phenyl)-1-phenylvinyl)styryl)-1-methylpyridin-1-ium

(TPEPy-

TEG, Figure 1a), for tracking of individual zebrafish embryonic cell proliferation and differentiation with bright signals. TPEPy-TEG is derived from a water-insoluble AIEgen, TPEPy. Because of the introduced hydrophilic TEG arms, TPEPy-TEG shows good water-solubility, which makes it difficult to pass through the cytoplasm membrane through endocytic pathways.


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Meanwhile, the TEG arms help to increase molecular weight, and the positive charge endows TPEPy-TEG low lipophilicity. All the factors are severely against the Lipinski’s “Rule of 5” for passive permeation to cell membrane, making TPEPy-TEG difficult to pass through the cytoplasm membrane through passive diffusion.28 Therefore, the two hydrophilic TEG arms were conjugated to TPEPy to endow it with poor cell permeability. In fact, this approach mitigates the diffusion of small-molecular-weight TPEPy-TEG to neighboring cells during the whole process of injectionbased lineage tracing. After injection, electrostatic interaction between positively charged pyridinium and intracellular biomolecules can activate AIE of TPEPy-TEG and provides strong signals against the dilution of cell division.

Figure 1. a) Chemical structures of TPEPy and TPEPy-TEG; b) Absorption spectrum of TPEPyTEG in water (10 μM) and emission spectra of TPEPy-TEG in water (10 μM) and solid state, λex


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= 405 nm; c) Relative emission intensities of TPEPy-TEG after incubation in PBS or with SDS (20 μM), BSA (0.01 mg/mL), DNA (0.3 μM), 3T3 cell lysate, (I0 represents the intensity in water, I represents the intensity in different condition, λex = 405 nm); Confocal and bright-field images of 3T3 cells treated with TPEPy (10 μM) (d) and TPEPy-TEG (10 μM) (e). The excitation wavelength was 405 nm, and the emission was collected above 650 nm. RESULTS AND DISCUSSION The synthetic routes to TPEPy-TEG and TPEPy are shown in Scheme S1. In short, Heck coupling between compound 1 and 4-vinyl pyridine yielded compound 2; deprotection of methoxy groups in compound 2 yielded compound 3, which was further reacted with 2-(2-(2hydroxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate to obtain compound 4. TPEPy-TEG was finally produced by the reaction of compound 4 with iodomethane and ion exchange with silver hexafluorophosphate. TPEPy was similarly obtained by reaction of compound 2 with iodomethane and ion exchange with silver hexafluorophosphate. The chemical structures of all newly reported compounds were well characterized by nuclear magnetic resonance (Figures S1-3) and mass spectrometers. Before using TPEPy-TEG as a tracer for embryonic cell tracing, we first studied its photo properties in aqueous media. As shown in Figure 1b, benefitting from the donor-acceptor structure,29 the absorption of TPEPy-TEG in water is located in the visible region with a peak at 400 nm and the emission is located in the red/near-infrared region with a peak at 730 nm. The huge Stokes shift (330 nm) can minimize the interferences from self-absorption or auto-fluorescence. Meanwhile, the fluorescence intensity of TPEPy-TEG remained 97% upon continuous light excitation for 10 min, which is much more stable than that for fluorescein isothiocyanate (FITC) and rhodamine B (Figure S4), indicating good photostability of TPEPy-TEG.1 In addition, as compared to its emission in water, the emission of TPEPy-TEG in solid state is blue-shifted to 660


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nm and the intensity is enhanced by more than 6-fold, indicating the AIE-active characteristics of TPEPy-TEG. Although the emission of TPEPy-TEG is weak in phosphate buffered saline (PBS), it can be enhanced by several-fold upon interactions of TPEPy-TEG with negatively charged surfactant (SDS: sodium dodecyl sulfate) or biomolecules (BSA: bovine serum albumin, DNA: deoxyribonucleic acid, 3T3 cell lysate) (Figure 1c). TPEPy-TEG is composed of hydrophobic core, hydrophilic arms and positively charged pyridinium. Once incubated with the negatively charged surfactant, SDS, the strong electrostatic and hydrophobic interactions force them to form micronano assembly to inhibit the intramolecular motions of TPEPy-TEG and active AIE to enhance the emission.30 BSA is a nutrient protein with hydrophobic cavum and it is slightly negatively charged under physiological conditions. The emission of TPEPy-TEG can also be enhanced once it is captured in the hydrophobic cavum through electrostatic and hydrophobic interactions.31 DNA is a polynucleotide with many units of negatively charged phosphate groups. It mainly interacts with TPEPy-TEG via electrostatic interactions to activate the AIE property and enhance the emission.32 This property is highly important for cell lineage tracing because bright signals can be achieved after the injection of TPEPy-TEG into embryonic cells. The in vitro diffusion performance of TPEPy-TEG was then studied through its interactions with 3T3 cells. As can be seen in Figure S5, TPEPy formed relatively large nanoaggregates with an average hydrodynamic size of around 700 nm in PBS. Therefore, it could be efficiently taken up by 3T3 cells through endocytosis (Figure 1d).33 After modifying TPEPy with two neutral and hydrophilic TEG arms, the obtained TPEPy-TEG exhibits good water-solubility with an average hydrodynamic size of around 4 nm in PBS (Figure S5). As a result, no endocytosis occurred when TPEPy-TEG was incubated with live 3T3 cells (Figure 1e), which indicated poor cell membrane permeability. The same performance was also observed after incubation with HeLa and HEK-293


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cells (Figure S6). Nevertheless, the poor cell permeability cannot ensure that TPEPy-TEG would be entrapped in cells without leakage after being injected into target cells. To evaluate the probe leakage in cells directly, we have to send TPEPy-TEG into cells first. However, the cell membrane permeability of TPEPy-TEG is poor, and the small size of normal mammalian cells such as 3T3 cells makes it difficult for direction injection of cell trackers. Therefore, we modified TPEPy-TEG with cleavable acetyl groups to reduce its water-solubility, which enables the endocytosis of TPEPy-TEG-Ac into cells. Then the acetyl groups could be automatically cleaved in cells to generate TPEPy-TEG for cell tracking. In this regard, two acetyl-protecting groups were introduced to the two terminal hydroxyl groups (Scheme S1), yielding TPEPy-TEG-Ac (Figure 2a). As the water-solubility of TPEPy-TEG-Ac is much poorer than that of TPEPy-TEG, TPEPyTEG-Ac formed nanoaggregates with an average hydrodynamic size of around 50 nm in PBS (Figure S7), which could be taken up by living 3T3 cells through endocytosis. The complete hydrolysis of the protecting esters in 3T3 cells was first verified through high-performance liquid chromatography (HPLC) analysis. 3T3 cell lysate was used to hydrolyze esters in TPEPy-TEGAc to estimate the time required for complete hydrolysis of the protecting esters. As shown in Figure S8, TPEPy-TEG-Ac has a retention time of 13.3 min in HPLC analysis. After incubation with 3T3 cell lysate in PBS at 37 ℃ for 24 h, the peak at 13.3 min nearly disappeared and a new strong peak at 10.4 min arose, which was similar to the retention time of TPEPy-TEG. This indicates the nearly complete transformation of TPEPy-TEG-Ac to TPEPy-TEG. Therefore, it was hypothesized that TPEPy-TEG-Ac would be completely transformed to TPEPy-TEG in 3T3 cells within 24 h. As a consequence, the diffusion performance of TPEPy-TEG was evaluated through the following steps: (i) after incubation of TPEPy-TEG-Ac with 3T3 cells for 30 min, the free TPEPy-TEG-Ac was washed out; (ii) the 3T3 cells pre-treated with TPEPy-TEG-Ac were then


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cultured for 24 h to ensure a complete ester hydrolysis; (iii) fresh 3T3 cells were co-incubated with the pre-treated 3T3 cells for another 24 h to examine the presence of any diffused tracers in the newly added cells. After all these treatments, strong signal was only observed in the TPEPy-TEGAc-pre-treated 3T3 cells, while no signal was observed in the newly added cells (Figure 2b), indicating that the intracellular TPEPy-TEG was entrapped within the cells and it could not diffuse to the neighboring cells. This result indicates that TPEPy-TEG-Ac can be used for cell group lineage tracing without leakage. Moreover, obvious red signal could still be observed from the TPEPy-TEG-Ac-pre-treated 3T3 cells after a 6-day incubation (Figure 2c). This long-term cellular tracing capability is ascribed to the complete entrapment of generated TPEPy-TEG within 3T3 cells and their AIE characteristics upon binding to different biomolecules inside cells. In addition, TPEPy-TEG, TPEPy-TEG-Ac and TPEPy all exhibited good biocompatibility towards 3T3 cells even at high concentrations (Figure S9). Particularly, 3T3 cells still maintained a high viability of 90% after incubation with 64 μM of TPEPy-TEG.


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Figure 2. a) Acetyl cleavage reaction of TPEPy-TEG-Ac to produce TPEPy-TEG upon esterase treatment; b) Confocal and bright-field images of 3T3 cells pre-treated with TPEPy-TEG-Ac (10 μM) co-cultured with unstained 3T3 cells for 24 h; c) Confocal and bright-field images of 3T3 cells at designated days after labelled by TPEPy-TEG-Ac (10 μM). Concentration of cell: 60,000 per well. The excitation wavelength was 405 nm, and the emission was collected above 650 nm. After confirming the long-term tracking ability and the total entrapment of TPEPy-TEG in 3T3 cells, TPEPy-TEG was utilized for micro-injection-based embryonic cell tracing in zebrafish model. Zebrafish is a vertebrate model which is widely used in cell and developmental biology. It enables the investigation of various biological phenomena at cellular and sub-cellular resolutions. In general, the zebrafish embryos transition from the single-cell stage to the mid-blastula stage based on deterministic maternal factors imparted by the zygotic genomic transcription.4 As seen in Figure 3a, once the zebrafish embryonic cell at 1-cell stage was injected with 30 pL of 300 μM


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TPEPy-TEG in ultrapure water for cell division tracing, from 1-cell stage to 8-cell stage, TPEPyTEG was evenly inherited by progenies of treated ancestor-cell, demonstrating its capacity in successful cell division tracing. To confirm that there was no TPEPy-TEG diffusion from the treated blastomere to another blastomere or yolk cell, single blastomere in the 4-cell stage was also injected with TPEPy-TEG. As seen in Figure 3b, along with the embryonic proliferation, TPEPyTEG was faithfully inherited by all cells in the lineage of the injected cells, and no transfer of label was observed in sister cells or yolk cell as the embryo transitioned from the 4-cell to the 32-cell stage.


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Figure 3. Images of zebrafish embryos injected with TPEPy-TEG (30 pL, 300 μM) in one cell at 1-cell (a) or 4-cell (b) stage and the proliferative embryo at 2-, 4-, 8-cell or 8-, 16-, 32-cell stage, respectively (left: bright field, right: overlay of confocal images with bright field). The labelled cells were circled with yellow colour, the unlabelled cells were circled with blue colour. The excitation wavelength was 405 nm, and the emission was collected above 650 nm. Subsequently, we injected zebrafish embryos with approximately 30-50 pL of TPEPy-TEG (300 µM) in the 64-cell cleavage stage to map the zebrafish fate (Figure 4a). The 64-cell cleavage stage of zebrafish embryo is at blastula stage. After the blastula stage, during gastrulation, the endoderm and mesoderm germ layers organize themselves to eventually form specific organs and tissues. This cascade of embryonic stages is found to be highly conserved throughout multiple vertebrate species. Therefore, injecting the embryo in a specific cohort of cells at different points in the germ layers can enable the visualization of fluorescent signals within specific organ systems, resulting in the identification of the mother blastomeres for different organs. As shown in Figure 4b, the injected embryos were imaged and screened using a fluorescent stereo microscope and were then allowed to grow to 24-28 h embryos. These embryos were subsequently imaged using confocal microscopy. Injection in the dorsal ectodermal region led to the appearance of fluorescent signal in the head, while the ectodermal injection in the ventral, mid-dorsal region, and animal pole region resulted in signals in the skin, hind-brain, and eye, respectively, which agrees well with the literatures3, 4 and indicates successful tracking of cell fate mapping in zebrafish. As time went on, the injected TPEPy-TEG was continuously diluted along with the proliferation and differentiation of embryonic cells. Interestingly, there was no significant reduction in the signal intensities in brain area as the treated embryo developed into zebrafish larvae in 30 h (Figure 5). These studies suggest that TPEPy-TEG is able to serve as a tracer for the injection-based lineage tracing with bright signals along with cell division-induced dye dilution.


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Figure 4. a) Scheme depicting zebrafish embryo with lineage label in different blastomeres for tissue-specific labelling; b) Fluorescent images of zebrafish embryos at 64-cell stage injected with 30-50 pL of TPEPy-TEG (300 µM) in dorsal, ventral, mid-dorsal, and animal pole regions, and confocal images with bright field of the respective 24-h embryos expressing red signal in head, skin, hind-brain and eye.


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Figure 5. Rainbow RGB images (ImageJ) of zebrafish embryo injected with TPEPy-TEG (30 pL, 300 μM) at 64-cell stage, then proliferation and differentiation for 0, 12, 20, and 30 h, respectively (right fourth picture is the enlarged image of the lower one). To further confirm the specific tissue localization, transgenic zebrafish line expressing skin specific green fluorescent protein (GFP) was chosen. Tg(cldnB:lynEGFP) zebrafish embryo neuromasts and skin cells express GFP and can serve as an ideal tool to visualize skin cell localization of the lineage label.34 Therefore, Tg(cldnB:lynEGFP) embryos were injected with TPEPy-TEG in the ventral ectodermal region (Figure 6a). As expected, the red signals from TPEPy-TEG localized on the same plane as GFP in the skin cells (Figure 6b). In the enlarged images (Figure 6c and 6d), it is clear to see that the red signal from TPEPy-TEG labels intracellular components while the green signal from GFP labels the surface of skin cells, and they both spread throughout the epidermis of the 24-hr embryo. The movie in supporting information presents the z-stack of images confirming the red signal localization in the same plane as the GFP signal and the stack of images were further evaluated through 3D projection in Image J.


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Figure 6. a) Fluorescence images of Tg(-8.0cldnb:lynEGFP) zebrafish embryos at 64-cell stage injected with 30-50 pL of TPEPy-TEG (300 µM) in ventral regions; b) image of 24-hr pretreated embryo (overlay of GFP channel, TPEPy-TEG and bright field, 3D projection in ImageJ); (c, d) Enlarged images of the 24-hr pretreated embryo. TPEPy-TEG: the excitation wavelength was 405 nm, and the emission was collected above 650 nm. GFP: the excitation wavelength was 488 nm, and the emission was collected 505-525 nm. CONCLUSION In summary, we have successfully designed and synthesized a small-molecular-weight AIEgen, TPEPy-TEG, for lineage tracking with bright signals. TPEPy-TEG possesses advantageous photo properties highly attractive for intracellular tracing, which include large Stokes shift of 330 nm in water, high photostability and good biocompatibility. More importantly, the two hydrophilic TEG arms endow TPEPy-TEG with poor cell permeability. After being injected into ancestor cells, TPEPy-TEG could be entrapped in the cells without leakage, enabling


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specific tracking of progenies of the treated ancestor cell to realize lineage tracing. The small molecule of TPEPy-TEG is evenly distributed in daughter cells, which favors lineage tracing with bright signals. Contradictory to traditional lineage tracers, which face the problem of low operating concentration and thus fast decreased signals along with rapid cell division, AIE-based TPEPyTEG can work at high concentrations and provide bright signals during lineage tracing. As this AIEgen tracer has several advantages as compared to the commonly used tracers, such as HRP and fluorogen-dextran conjugate, it is therefore promising for the development of lineage tracing techniques. EXPERIMENTAL SECTION Zebrafish Line. Four pairs of wild type or cldnb:lyn transgenic zebrafish were crossed by spawning overnight. Post fertilization, newly formed embryos settled to the tank bottom, and the water was decanted to collect the embryo through a sieve. The healthy embryos were incubated at 27 oC, 0.4% CO2 and raised in egg water (10% NaCl; 1.63% MgSO4·7H2O; 0.4% CaCl2; 0.3% KCl). Zebrafish Microinjection. Micro-injection of TPEPy-TEG into 4-cell/64-cell stage zebrafish embryos was done using a Harvard Apparatus nitrogen gas injector. The glass capillary needle used for injection was pulled through a heating element (O.D. 1.0 mm, I.D. 0.75 mm) in a needle puller into fine needles (15 µm). A 300 µM solution of TPEPy-TEG was filled into the needles which were loaded into the nitrogen gas injector. The injector was operated in pulse mode at 0.5 psi for the intra-cellular delivery of TPEPy-TEG. The embryos were placed against a glass slide in petri-dish for embryo injection and mounted in glass bottom dishes in 5% methyl cellulose for the confocal imaging (Carl Zeiss LSM 800); for TPEPy-TEG detection, the excitation wavelength was 405 nm, and the emission was collected above 650 nm; for GFP detection, the excitation wavelength was 488 nm, and the emission filter was 505-525 nm. The images were analyzed by Image J 1.43 × program (http://rsbweb.nih.gov/ij/).


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Supporting Information. Synthesis and structure identification data of TPEPy, TPEPy-TEG and TPEPy-TEG-Ac, photostability of TPEPy-TEG, FITC and Rhodamine B, dynamic light scattering size distribution and biocompatibility study of TPEPy, TPEPy-TEG and TPEPy-TEG-Ac, HPLC analysis of TPEPy-TEG-Ac, 3T3 cell lysate incubated TPEPy-TEG-Ac (24 h at 37 ℃) and TPEPyTEG. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Author Contributions #

These authors contributed equally to this work.

ACKNOWLEDGMENT We thank Singapore NRF Competitive Research Program (R279-000-483-281), NRF Investigatorship (R279-000-444-281), National University of Singapore (R279-000-482-133) for financial support. REFERENCES 1.

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For Table of Contents Use Only

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Visualize Embryogenesis and Cell Fate Using ... - ACS Publications

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