Complex 3D Structures Research Articles

CuCrZr Alloy Manufactured by LPBF Process: Correlation Between Microstructure, Mechanical and Thermal Properties

Copper based alloys are promising materials for electrical and thermal devices. In this respect, the use of laser powder bed fusion (LPBF) allows the realization of 3D complex structures, possibly enhancing the functional performances of such devices. LPBF also induces refined microstructures and build-up of residual stresses, due to the rapid solidifications and high cooling rate. In this work a CuCrZr alloy was manufactured by LPBF and a process feasibility window was defined. The effect of a heat treatment, consisting of solution annealing and artificial aging, on the microstructure, tensile and thermal properties was evaluated. Moreover, particular attention was addressed to the effect of the building direction on the thermal behaviour.It was found that thermal conductivity is improved by the heat treatment and that thermal conductivity resulted anisotropic, being higher in the growth direction.

Topological data analysis expands the genotype to phenotype map for 3D maize root system architecture.

A central goal of biology is to understand how genetic variation produces phenotypic variation, which has been described as a genotype to phenotype (G to P) map. The plant form is continuously shaped by intrinsic developmental and extrinsic environmental inputs, and therefore plant phenomes are highly multivariate and require comprehensive approaches to fully quantify. Yet a common assumption in plant phenotyping efforts is that a few pre-selected measurements can adequately describe the relevant phenome space. Our poor understanding of the genetic basis of root system architecture is at least partially a result of this incongruence. Root systems are complex 3D structures that are most often studied as 2D representations measured with relatively simple univariate traits. In prior work, we showed that persistent homology, a topological data analysis method that does not pre-suppose the salient features of the data, could expand the phenotypic trait space and identify new G to P relations from a commonly used 2D root phenotyping platform. Here we extend the work to entire 3D root system architectures of maize seedlings from a mapping population that was designed to understand the genetic basis of maize-nitrogen relations. Using a panel of 84 univariate traits, persistent homology methods developed for 3D branching, and multivariate vectors of the collective trait space, we found that each method captures distinct information about root system variation as evidenced by the majority of non-overlapping QTL, and hence that root phenotypic trait space is not easily exhausted. The work offers a data-driven method for assessing 3D root structure and highlights the importance of non-canonical phenotypes for more accurate representations of the G to P map.

Tuning thermoresponsive properties of carboxymethyl cellulose (CMC)–agarose composite bioinks to fabricate complex 3D constructs for regenerative medicine

3D bioprinting has emerged as a viable tool to fabricate 3D tissue constructs with high precision using various bioinks which offer instantaneous gelation, shape fidelity, and cytocompatibility. Among various bioinks, cellulose is the most abundantly available natural polymer & widely used as bioink for 3D bioprinting applications. To mitigate the demanding crosslinking needs of cellulose, it is frequently chemically modified or blended with other polymers to develop stable hydrogels. In this study, we have developed a thermoresponsive, composite bioink using carboxymethyl cellulose (CMC) and agarose in different ratios (9:1, 8:2, 7:3, 6:4, and 5:5). Among the tested combinations, the 5:5 ratio showed better gel formation at 37 °C and were further characterized for physicochemical properties. Cytocompatibility was assessed by in vitro extract cytotoxicity assay (ISO 10993–5) using skin fibroblasts cells. CMC-agarose (5:5) bioink was successfully used to fabricate complex 3D structures through extrusion bioprinting and maintained over 80 % cell viability over seven days. Finally, in vivo studies using rat full-thickness wounds showed the potential of CMC-agarose bulk and bioprinted gels in promoting skin regeneration. These results indicate the cytocompatibility and suitability of CMC–agarose bioinks for tissue engineering and 3D bioprinting applications.

Review of: "nanopatterns and complex 3D structures, as well as the aspect The above characteristics of the ratio regarding these outstanding advantages have also resulted"

Review of: "nanopatterns and complex 3D structures, as well as the aspect The above characteristics of the ratio regarding these outstanding advantages have also resulted"

Research progress of 3D-bioprinted functional pancreas and in vitro tumor models

With the rapid development of three-dimensional (3D) bioprinting technology, the research revolving around in vitro functional pancreas and tumor models has become the focus of attention in the field of life sciences. This review aims to summarize and deeply discuss the research progress and prospects of 3D-bioprinted functional pancreas and in vitro tumor models. The efforts in improving 3D printing technology to increase its accuracy and reliability in the biomedical applications have been ramped up over the past few years. Researchers are now able to create highly complex 3D structures through precise layering of biological materials at the micron scale. For instance, a functional pancreas can be printed in vitro by combining cells, biomaterials, and growth factors. The introduction of new technologies allows researchers to more accurately simulate the growth and spread of tumors, providing a more realistic platform for cancer treatment research. This not only helps accelerate the process of drug screening, but also lays the foundation for personalized medicine. As multiple disciplines, such as materials science, cell biology, and engineering, continue to converge with 3D bioprinting, emergence of more innovative applications is anticipated. However, despite significant progress, many technical and ethical challenges still need to be overcome before practical clinical applications can be implemented. In summary, the application of bioprinting technology is of great significance to the study of functional pancreas and in vitro tumor models, which could lead to new breakthroughs in the development of clinical treatment and personalized medicine.

Unleashing the Power of Undifferentiated Induced Pluripotent Stem Cell Bioprinting: Current Progress and Future Prospects.

Induced pluripotent stem cell (iPSC) technology has revolutionized various fields, including stem cell research, disease modeling, and regenerative medicine. The evolution of iPSC-based models has transitioned from conventional two-dimensional systems to more physiologically relevant three-dimensional (3D) models such as spheroids and organoids. Nonetheless, there still remain challenges including limitations in creating complex 3D tissue geometry and structures, the emergence of necrotic core in existing 3D models, and limited scalability and reproducibility. 3D bioprinting has emerged as a revolutionary technology that can facilitate the development of complex 3D tissues and organs with high scalability and reproducibility. This innovative approach has the potential to effectively bridge the gap between conventional iPSC models and complex 3D tissues in vivo. This review focuses on current trends and advancements in the bioprinting of iPSCs. Specifically, it covers the fundamental concepts and techniques of bioprinting and bioink design, reviews recent progress in iPSC bioprinting research with a specific focus on bioprinting undifferentiated iPSCs, and concludes by discussing existing limitations and future prospects.

A comprehensive study on the jet electrodeposition method for fabricating 3D structures with overhangs

Electrochemical additive manufacturing (ECAM) is gaining increasing attention in the fabrication of micro/nano-scale metal structures due to its advantages, such as high forming accuracy, low porosity, absence of thermal stress, etc. Among various ECAM techniques, interest is increasingly enhanced in jet electrochemical deposition (Jet ECD) approach due to its ultra-high deposition rate. Despite its inherent advantages as a high-efficiency ECAM process, Jet ECD still faces challenges for automated fabrication of complex 3D structures due to suitable control of the jetted flow. In the present study, a step-delay method based on Jet ECD is proposed. This approach allows for the automation of the fabrication process using commonly employed stepper motor translational stages, seamlessly integrating it into the current 3D printing framework. By adjusting the moving time of the stages and the delay time between two successive steps, agreement between the movement of the stages and the temporally nonlinear electrodeposition rate can be achieved. Consequently, complex 3D copper structures with overhangs, such as 3D helical structures, are successfully obtained. It is found the deposition rate can be as high as ∼25 µm/min, and the maximum overhanging angle that can be achieved by the automated step-delay method is ∼60°. The mechanical properties of the deposited copper using the proposed approach have been thoroughly characterized by various techniques. The average grain size of the deposited copper can be as small as 0.5 µm without preferred orientation. It also shows that highly dense structures with porosity of less than 0.1% can be obtained, and the elastic modulus of the deposited copper ranges from 3 - 20 GPa. In future studies, it is anticipated that larger overhanging angles can be achieved by incorporating image recognition techniques in the step-delay method.

Analysis of spontaneous ignition of hydrogen-enriched methane in a rectangular tube

This study investigates the spontaneous ignition of high-pressure hydrogen-enriched methane in air within a rectangular tube. A computationally efficient approach has been adopted, utilizing a reduced reaction mechanism and ignition delay model within a 3D Large Eddy Simulation (LES) framework. This approach overcomes the limitations of traditional 1D and 2D simulations with detailed chemistry models, which are unable to accurately reproduce the complex 3D shock wave structures within the tube. The simulated shock wave behavior during 9 MPa hydrogen leakage (case 1) and 11 MPa 90 vol% hydrogen/10 vol% methane mixture leakage (case 2) are found to agree well with experimental observations. In case 2, the hot spots generated by reflected shock waves and Mach reflections ignite the hydrogen/methane-air mixture, resulting in three sequential spontaneous ignitions. The flame is observed to primarily propagate along the tube corners and wall centers, with the central ignition spreading across the entire cross-section. For the 25 MPa 24 vol% hydrogen/76 vol% methane mixture leakage (case 6), the shock intensity is significantly reduced due to the increased methane proportion, leading to spontaneous ignition only at the tube corners when the hemispherical shock wave reflects from the wall. The flame predominantly forms downstream along the tube corner, gradually spreading along the tube wall. It is indicated that while the probability of spontaneous ignition decreases with increasing methane content, the risk remains significant under sufficiently high pressures. To the best our knowledge, this study represents the first 3D large eddy simulation of spontaneous ignition for high-pressure hydrogen-enriched methane leakage into air, providing valuable insights into the underlying physical phenomena.

Manufacturing seamless three-dimensional woven preforms with complex shapes based on a new weaving technology

A new weaving technology using a modified z-binder interlacement system was designed to demonstrate its potential for the effective, continuous, efficient, and rapid manufacturing of various three-dimensional (3D) woven structures. First, three representative 3D woven preforms were fabricated. Then, epoxy resin was transferred to a preform. The manufactured 3D woven textile-reinforced composites were investigated using micro-CT analysis, tensile tests, and bending tests to study the effect of the z-binder interlacing on the structure. Furthermore, a design rule was established that could seamlessly create complex 3D woven structures with non-uniform heights in the z-direction, such as boxes, bowls, and pyramids, demonstrating that the seamless 3D woven preform of the complex shape can be fabricated with structural integrity.

3D printable and mechanically tunable hydrogels achieved through hydrophobic and ionic interactions.

Thermal stiffening materials are commonly applied in the aerospace and automotive industries, among others, since their dimensional stabilities and stiffness characteristics improve at high temperatures. In this study, temperature-triggered modulus-tunable hydrogels were prepared by combining Pluronic F-127 with charged polymers. Pluronic F-127, a triblock copolymer micelle, provided three-dimensional printing capabilities of fine resolution with high viscosity, while hydrophobic and ionic interactions among polymer networks provided thermal stiffening. The hydrogel ink's printability was demonstrated by successfully creating complex 3D structures. A calcium ion carrying a hydrophobic propionate and carboxylate group in polymer chains was used to form additional physical crosslinking at high temperature, ultimately leading to the thermal stiffening effect without volume change. The thermal stiffening behavior was found to be fully reversible and repeatable. Finally, to demonstrate the versatility of this work, graphene oxide was added to produce a light-controllable modulus based on its photothermal properties.

Supercritical CO2 Assisted Ni-P Electroless Plating of PET Components with Complex 3D Structures

There is an increasing demand of methodologies to metallize complex polymer materials for application in electronics such as wearable devices and robotic products. In various metallization methods, electroless plating is a powerful and promising approach to meet this demand. The present conventional electroless plating requires pretreatment steps to deposit catalyst onto surfaces of polymer materials, which results in damages to their surfaces and also deformation of their shape because of the usage of strong acidic solvents. Furthermore, the adhesion strength between the polymer material and the metal layer is insufficient. To overcome these problems, a catalyzation step using supercritical carbon dioxide (sc-CO2) as the solvent is proposed.[1] The sc-CO2-assisted catalyzation enables a deep impregnation of catalysts into the polymer material without the pretreatment step, and this enhances the adhesive properties of the deposited metal on the polymer. By the sc-CO2-assisted catalyzation, electroless Ni-P plating of polyethylene terephthalate (PET) is realized. [1] Metallization of polymer materials with complex 3D structures is a more attractive topic, but it is widely accepted to be more difficult than film structures. Also, application the electroless plating via sc-CO2-asssited catalyzation to 3D-structured polymer materials remains unexplored. In this presentation, we report Ni-P plating of PET samples having complex 3D structures by the sc-CO2-assisited catalyzation process.A PET based material with a concavo-convex structure was designed and fabricated by the processing machine as shown in Figs. 1(a)–(b). Sc-CO2-assisted catalyzation process was performed in a high pressure reactor[1] containing palladium (II) hexafluoroacetylacetonate (Pd(C5HF6O2)2) as the palladium catalyst source at 70 °C under 15 MPa for 2 h. After the catalyzation process, the reactor was cooled to room temperature while maintaining the pressure in the reactor, and then the pressure in the reactor was reduced to atmospheric pressure. The collected PET sample from the reactor was kept under ambient atmosphere for two weeks, and then it was subjected to electroless Ni-P plating at 70 ℃ for 10 min.Fig. 1(c) shows the PET sample obtained after the sc-CO2-assisted catalyzation process followed by the post-treatment process. The volume of the catalyzed PET sample was scarcely changed. On the other hand, the post-treatment processes performed under the different conditions were found to cause undesired swelling and white turbidity of the PET sample, presumably because of microbubbles derived from residual CO2 in the PET. Fig. 1(d) shows an image of Ni-P successfully metallized on the PET surface. The electrical resistance of the Ni-P/PET material was 8.7 Ω, which is acceptable for electronic device applications. From these results, we concluded that sc-CO2-assisted catalyzation is applicable to metallization of PET materials with complex 3D structures. Reference [1] P.-W. Cheng, C.-Y. Chen, T. Ichibayashi, T.-F.M. Chang, M. Sone, S. Nishimura, J. Supercrit. Fluids 180 (2022) 105455. Figure 1

Sacrificial-Rotating Rod-Based 3D Bioprinting Technique for the Development of an In Vitro Cardiovascular Model.

Several studies have attempted to develop complex cardiovascular models, but the use of multiple cell types and poor cell alignments after fabrication have limited the practical application of these models. Among various bioprinting methods, extrusion-based bioprinting is the most widely used in the bioengineering field. This method not only has the potential to construct complex 3D biological structures but it also enables the alignment of cells in the printing direction owing to the application of shear stress to the cells during the printing process. Therefore, this study developed an in vitro cardiovascular model using an extrusion-based bioprinting method that utilizes a rotating rod as a printing platform. The rotating rod was made of polyvinyl alcohol (PVA) and used as a sacrificial rod. This rotating platform approach enabled the printing of longer tubular-vascular structures of multiple shapes, including disease models, and the water-soluble properties of PVA facilitated the isolation of the printed vascular models. In addition, this method enabled the printing of the endothelial cells in the bloodstream direction and smooth muscle cells in the circumferential direction to better mimic the anatomy of real blood vessels. Consequently, a cardiovascular model was successfully printed using a gelatin methacryloyl bioink with cells. In conclusion, the proposed fabrication method can facilitate the fabrication of various cardiovascular models that mimic the alignment of real blood vessels.

Robust Superhydrophobicity through Surface Defects from Laser Powder Bed Fusion Additive Manufacturing.

The robustness of superhydrophobic objects conflicts with both the inevitable introduction of fragile micro/nanoscale surfaces and three-dimensional (3D) complex structures. The popular metal 3D printing technology can manufacture robust metal 3D complex components, but the hydrophily and mass surface defects restrict its diverse application. Herein, we proposed a strategy that takes the inherent ridges and grooves' surface defects from laser powder bed fusion additive manufacturing (LPBF-AM), a metal 3D printing process, as storage spaces for hydrophobic silica (HS) nanoparticles to obtain superhydrophobic capacity and superior robustness. The HS nanoparticles stored in the grooves among the laser-melted tracks serve as the hydrophobic guests, while the ridges' metal network provides the mechanical strength, leading to robust superhydrophobic objects with desired 3D structures. Moreover, HS nanoparticles coated on the LPBF-AM-printed surface can inhibit corrosion behavior caused by surface defects. It was found that LPBF-AM-printed objects with HS nanoparticles retained superior hydrophobicity after 150 abrasion cycles (~12.5 KPa) or 50 cycles (~37.5 KPa). Furthermore, LPBF-AM-printed ships with superhydrophobic coating maintained great water repellency even after 10,000 cycles of seawater swashing, preventing dynamic corrosion upon surfaces. Our proposed strategy, therefore, provides a low-cost, highly efficient, and robust superhydrophobic coating, which is applicable to metal 3D architectures toward corrosion-resistant requirements.

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.

High-Resolution Additive Manufacturing of a Biodegradable Elastomer with A Low-Cost LCD 3D Printer.

Artificial organs and organs-on-a-chip (OoC) are of great clinical and scientific interest and have recently been made by additive manufacturing, but depend on, and benefit from, biocompatible, biodegradable, and soft materials. Poly(octamethylene maleate (anhydride) citrate (POMaC) meets these criteria and has gained popularity, and as in principle, it can be photocured and is amenable to vat-photopolymerization (VP) 3D printing, but only low-resolution structures have been produced so far. Here, a VP-POMaC ink is introduced and 3D printing of 80µm positive features and complex 3D structures is demonstrated using low-cost (≈US$300) liquid-crystal display (LCD) printers. The ink includes POMaC, a diluent and porogen additive to reduce viscosity within the range of VP, and a crosslinker to speed up reaction kinetics. The mechanical properties of the cured ink are tuned to match the elastic moduli of different tissues simply by varying the porogen concentration. The biocompatibility is assessed by cell culture which yielded 80% viability and the potential for tissue engineering illustrated with a 3D-printed gyroid seeded with cells. VP-POMaC and low-cost LCD printers make the additive manufacturing of high resolution, elastomeric, and biodegradable constructs widely accessible, paving the way for a myriad of applications in tissue engineering and 3D cell culture as demonstrated here, and possibly in OoC, implants, wearables, and soft robotics.

Development of heart organoid cryopreservation method through Fe3 O4 nanoparticles based nanowarming system.

Beyond single cell two-dimensional (2D) culture, research on organoids that can mimic human organs is rapidly developing. However, there are still problems in commercialization and joint research using organoids due to the lack of technology to safely store organoids. Since organoids are 3D complex structures with a certain size (0.1-5mm) beyond the size of cells, the conventional cell-level cryopreservation method using cryoprotectant (CPA) cannot overcome the damage caused by volume change due to osmotic pressure difference and ice nucleation. Herein, we attempted to solve such limitations by applying a nanowarming system using CPA with high cell permeability and Fe3 O4 nanoparticles. By performing beat rate measurement, histological analysis, contractility analysis, and multi-electrode array, it was verified that the developed method could significantly improve functional recovery and survival of heart organoids after freezing and thawing. In this study, we demonstrated a successful organoid cryopreservation method based on a Fe3 O4 nanowarming system. The developed technology will provide clues to the field of tissue cryopreservation and spur the application of organoids.

Multishape Programming of Shape Memory Polymer Assemblies Fabricated by Vat Photopolymerization-Based 3D Printing and Interfacial Welding.

The combination of three-dimensional (3D) printing and shape memory polymers (SMP) enables programmable shape morphing of complex 3D structures, which is commonly termed four-dimensional (4D) printing. The process requirements of vat photopolymerization-based 3D printing impose limitations on the molecular structure design of SMPs, making it challenging to achieve triple- or multiple-shaped memory effects. Herein, we printed SMPs with different Tg values and obtained an SMP assembly through interfacial welding. The welding process is facilitated by the dynamic exchange of hindered urethane bonds at the interface. The resulting SMP assembly exhibits a quadruple shape memory effect, enabling programmable sequential deformation. The advantage of this approach is that the molecular design and the corresponding thermodynamic properties of different welding SMP components can be independently adjusted, enabling a greater range of shape and functional variations in the final 3D SMP assembly.

Review of research progress and development trend of digital image correlation

PurposeThe purpose of this paper is to provide a comprehensive review of a non-contact full-field optical measurement technique known as digital image correlation (DIC).Design/methodology/approachThe approach of this review paper is to introduce the research pertaining to DIC. It comprehensively covers crucial facets including its principles, historical development, core challenges, current research status and practical applications. Additionally, it delves into unresolved issues and outlines future research objectives.FindingsThe findings of this review encompass essential aspects of DIC, including core issues like the subpixel registration algorithm, camera calibration, measurement of surface deformation in 3D complex structures and applications in ultra-high-temperature settings. Additionally, the review presents the prevailing strategies for addressing these challenges, the most recent advancements in DIC applications across quasi-static, dynamic, ultra-high-temperature, large-scale and micro-scale engineering domains, along with key directions for future research endeavors.Originality/valueThis review holds a substantial value as it furnishes a comprehensive and in-depth introduction to DIC, while also spotlighting its prospective applications.

Fabrication of microneedles using two photon-polymerization with low numerical aperture

Three-dimensional (3D) micro-nano fabrication through two-photon polymerization (TPP) is a prominent approach for fabricating complex 3D structures. However, the existing techniques face limitations, such as small fabrication area, dependence on high numerical aperture (e.g., oil immersion) objective lenses, which are more expensive. We developed a 780 nm fs laser system integrated with a 2D galvanometer system and Z axis to create a 3D micro-nano printing system. A method to print arbitrarily complex 3D structures while maintaining an extremely high lateral spatial resolution of sub-100nm is proposed by using a cheap low numerical aperture objective lens with the laser power and exposure time precisely controlled. The impact of the laser power and exposure time on the resolution of 3D micro-nano processing is investigated, and we identify the optimal processing environment for achieving the best resolution. The printed linewidth of 62 nm was achieved without the use of an oil immersed objective lens, and only one laser beam was used instead of stimulated emission depletion. Microneedle arrays for animal injection were fabricated using this strategy, which can improve the performance of animal organoid repair. Our results suggest that TPP micro-nano fabrication is an effective method for processing microneedle arrays for biomedical engineering, with the ability to improve the processing resolution with a cheap low numerical aperture objectives lens (e.g., NA = 0.4) through meticulous control of laser power and exposure time. This approach opens up possibilities for affordable and scalable fabrication in sub-100nm structures.

Adaptive Optics Microscopy with Wavefront Sensing Based onNeighbor Correlation.

Complex structures in living cells and tissues induce wavefront errors when light waves pass through them, and images observed with optical microscopes are undesirably blurred. This problem is especially serious for living plant cells because images are strikingly degraded even within a single cell. Adaptive optics (AO) is expected to be a solution to this problem by correcting such wavefront errors, thus enabling high-resolution imaging. In particular, scene-based AO involves wavefront sensing based on the image correlation between subapertures in a Shack-Hartmann wavefront sensor and thus does not require an intense point light source. However, the complex 3D structures of living cells often cause low correlation between subimages, leading to loss of accuracy in wavefront sensing. This paper proposes a novel method for scene-based sensing using only image correlations between adjacent subapertures. The method can minimize changes between subimages to be correlated and thus prevent inaccuracy in phase estimation. Using an artificial test target mimicking the optical properties of a layer of living plant cells, an imaging performance with a Strehl ratio of approximately 0.5 was confirmed. Upon observation of chloroplast autofluorescence inside living leaf cells of the moss Physcomitrium patens, recovered resolution images were successfully obtained even with complex biological structures. Under bright-field illumination, the proposed method outperformed the conventional method, demonstrating the future potential of this method for label- and damage-free AO microscopy. Several points for improvement in terms of the effect of AO correction are discussed.