**3. Results and Discussion**

#### *3.1. Fabricating Hollow 3D Structures with Selected Spatial Resolution*

By laser scanning inside the photoresist (Figure 2b), the smart properties of materials provided fundamental responsiveness as beneficial advantages in devices. For example, the hydrogel-based microlenses (Figure 3a) exhibited the ability to change light facula similar to a dynamic focus lens (Video S2). Seen in Figure 3a, the diameter of the lens changed from about 20 µm to 29 µm, meaning the swelling area in the X–Y plane increased by at least 100% (seen in the Video S3). Subsequently, we changed the volume of the hydrogels and measured the volume alternation to confirm an approximate swelling ratio of >210% (seen in Figure S5). Moreover, the as-prepared hydrogel inherited the pH-responsive ability such as the previously studied bio-materials [6,45,46]. By slowly changing the pH value to an acidic environment, the in-solution hydrogel further swelled and stretched itself out (seen in Video S4). With the micro/nanoscale deformable structures, these smart devices promise broadband applications for embedding bio-conditions.

The experimental observation confirmed the desirable mobility of the photoresist and highly effective two-photon absorption at a mild laser power. The photon-induced cross-linking reaction was confined at the submicron voxel (Figure S5). All complex 3D scaffolds self-stood on substrates in the absence of supportive tools. Both the minimum line width and minimum height could reach 150 nm (Figure S6). The experiment generally concluded a suitable scanning speed from 30 to 140 µm/s with an average optical power distributed from 2 to around 20 mW. To check the formation quality for complex hollow structures widely applied for cell proliferation [9], micro machinery [47] or microfluidic [48], a batch of scaffolds consisting of specific tetrahedrons and cubes was firstly fabricated and characterized (Figure 3b,e).

An ultrafine feature size was observable in the CCD or SEM images (tetrahedron in Figure 3b,c, cubes in Figure 3d,e). By adjusting the scanning speed, the equivalent power exposure dose affected the volume of the cross-linking degree a lot. For example, the spatial resolution of Figure 3b scanned at 10 µm/s was better than that of Figure 3c scanned at 1 µm/s. The same selective ability was manifested again in the shape of the cubes. A higher exposure dose of the pulsed laser beam triggered a higher-level polymerization. Based on this factor, the optical parameters could determine the feature size.

Furthermore, the spectrometer-reconstructed fluorescence images of structures (Figure 3c,e) matched well with the SEM images. The highlighted fluorescence implied that the freestanding hollow structures used pure organic hydrogel materials without hard metal. Due to the bio-compatibility, desirable adhesion, and structural complexity of HAMA, and PEG-da [31], the demonstrated scaffolds promised more practical cell

*Micromachines* **2022**, *13*, x FOR PEER REVIEW 6 of 13

ported on soon.

applications. The tests of cell viability and adhesion of hydrogels are ongoing, and will be reported on soon. the use of small voxels and a tight arrangement. All the demonstrated hydrogels here closely followed the design, although not ideally, as some geometric variations resulted from the intrinsic material properties, development, and observation method.

HAMA, and PEG-da [31], the demonstrated scaffolds promised more practical cell applications. The tests of cell viability and adhesion of hydrogels are ongoing, and will be re-

Additionally, TPP is typically an additive manufacturing process, where voxels stack up every layer, so the resolution of the laser voxels also plays a vital role in precise control. A smaller laser voxel determines a better spatial arrangement in the same exposure dose. By changing the magnifying ability of objectives, for example, N.A. from 1.2 to 1.43, the fine voxel generated an ultrafine resolution. As compared in Figure 3d,e, samples were scanned with N.A. of objectives = 1.43 and 1.2, respectively. The high accuracy implied

**Figure 3.** (**a**) A group of dynamic focus lenses using the proposed hydrogel materials, which tuned the size of facula by swelling-to-shrinkage on background light in a dark room; additionally, a comparison of the single hydrogel lens before and after swelling is shown; (**b**–**e**) the fabricated bioscaffolds of tetrahedrons and cubes, respectively. (**b**) CCD, SEM, and zoomed-in SEM images of a triangle array of tetrahedrons is contained in the first panel; (**c**) SEM and fluorescence images (red) of tetrahedrons scanned at slow scanning speed about 5 μm/s; (**d**,**e**) observation of two arrays of cubes. Zoomed-in view shows a micrometer-level resolution (approximately 2 μm in (**d**), and 16 μm in (**e**)) to self-support the complex cube-stacked structures. In addition to the selective spatial resolution and biocompatibility, this 532 nm TPP **Figure 3.** (**a**) A group of dynamic focus lenses using the proposed hydrogel materials, which tuned the size of facula by swelling-to-shrinkage on background light in a dark room; additionally, a comparison of the single hydrogel lens before and after swelling is shown; (**b**–**e**) the fabricated bio-scaffolds of tetrahedrons and cubes, respectively. (**b**) CCD, SEM, and zoomed-in SEM images of a triangle array of tetrahedrons is contained in the first panel; (**c**) SEM and fluorescence images (red) of tetrahedrons scanned at slow scanning speed about 5 µm/s; (**d**,**e**) observation of two arrays of cubes. Zoomed-in view shows a micrometer-level resolution (approximately 2 µm in (**d**), and 16 µm in (**e**)) to self-support the complex cube-stacked structures.

utilized optical parameters and a material ratio to modulate the mechanical properties in a wide range. As tested using our previously reported micro-mechanics technique [10], a Young's modulus of as-prepared hydrogels presented a wide range from KPa to MPa Additionally, TPP is typically an additive manufacturing process, where voxels stack up every layer, so the resolution of the laser voxels also plays a vital role in precise control. A smaller laser voxel determines a better spatial arrangement in the same exposure dose. By changing the magnifying ability of objectives, for example, N.A. from 1.2 to 1.43, the fine voxel generated an ultrafine resolution. As compared in Figure 3d,e, samples were scanned with N.A. of objectives = 1.43 and 1.2, respectively. The high accuracy implied the use of small voxels and a tight arrangement. All the demonstrated hydrogels here closely followed the design, although not ideally, as some geometric variations resulted from the intrinsic material properties, development, and observation method.

In addition to the selective spatial resolution and biocompatibility, this 532 nm TPP utilized optical parameters and a material ratio to modulate the mechanical properties in a wide range. As tested using our previously reported micro-mechanics technique [10], a Young's modulus of as-prepared hydrogels presented a wide range from KPa to MPa

(Figure 4a), covering the general requirements from tissue engineering to mechanics. These tunable mechanics proved that hydrogels work as a structural material and functional material simultaneously. (Figure 4a), covering the general requirements from tissue engineering to mechanics. These tunable mechanics proved that hydrogels work as a structural material and functional material simultaneously.

**Figure 4.** (**a**) Young's modulus of hydrogels; (**b**) water reversely bent a planar hydrogel from the initial planar to a bent shape, and the scale bar is 50 μm; (**c**) ethanol bent a planar hydrogel to a bent shape as well, and the scale bar is 50 μm; (**d**) a spider-shaped hydrogel was fabricated using our smart materials and then scanned by a laser beam for local actuation, the area projected by laser absorbed photon energy, and thermally swelled to commence shape reconfiguration, and the scale bar is 50 μm; (**e**) the tadpole-shaped hydrogels stretched their tails by swelling, and the scale bar of CCD images is 50 μm. *3.2. Humidity and Light-Triggered Reverse Shape Morphing* **Figure 4.** (**a**) Young's modulus of hydrogels; (**b**) water reversely bent a planar hydrogel from the initial planar to a bent shape, and the scale bar is 50 µm; (**c**) ethanol bent a planar hydrogel to a bent shape as well, and the scale bar is 50 µm; (**d**) a spider-shaped hydrogel was fabricated using our smart materials and then scanned by a laser beam for local actuation, the area projected by laser absorbed photon energy, and thermally swelled to commence shape reconfiguration, and the scale bar is 50 µm; (**e**) the tadpole-shaped hydrogels stretched their tails by swelling, and the scale bar of CCD images is 50 µm.

#### Traditionally, researchers have often combined soft active hydrogels with hard inert *3.2. Humidity and Light-Triggered Reverse Shape Morphing*

materials in dual-layer designs for actuation in multi-step fabrication. Utilizing the different swelling-to-shrinkage degrees of different materials, subject to the environment, for example, the temperature results in self-folding machines. However, TPP incorporating smart hydrogels enables the macroscopic stationary structure to reach a micron-to-nanometer level 4D function using single materials in a single step. The molecular interactive force between the functional groups and applied stimuli (polar solvent, water, acid, or alkali solvent) contributed to the stimuli responsiveness. Therefore, we changed the optical power and spacing width formed during scanning to display the resilient shape deformation ability. Traditionally, researchers have often combined soft active hydrogels with hard inert materials in dual-layer designs for actuation in multi-step fabrication. Utilizing the different swelling-to-shrinkage degrees of different materials, subject to the environment, for example, the temperature results in self-folding machines. However, TPP incorporating smart hydrogels enables the macroscopic stationary structure to reach a micron-to-nanometer level 4D function using single materials in a single step. The molecular interactive force between the functional groups and applied stimuli (polar solvent, water, acid, or alkali solvent) contributed to the stimuli responsiveness. Therefore, we changed the optical power and spacing width formed during scanning to display the resilient shape deformation ability.

In nature, many plants use water sorption and desorption for motion or reversible shape morphing. To mimic this behavior at a microscale, we fabricated the water-swelling hydrogel. The polymeric matrix consisted of permanent covalent carbon bonds in polymer materials, and various chemical functional groups could collaborate with outside-In nature, many plants use water sorption and desorption for motion or reversible shape morphing. To mimic this behavior at a microscale, we fabricated the water-swelling hydrogel. The polymeric matrix consisted of permanent covalent carbon bonds in polymer materials, and various chemical functional groups could collaborate with outside-applied stimuli for judicious motion. As seen in Figure 4b,c, the humidity (or water) reversed the single-layer planar-like hydrogel, and the initial plane changed into a C type. No matter how frequently it is immersed into water or heated, the basic frame of the sample stayed unchanged, which denoted the cross-linked network's existence as a skeleton for structural integrity.

Here, the water molecules worked as the triggering condition, and the molecular force captured water to swell or shrink by heating to recover. The volume ratio shrank over 200% in evaporation, demonstrating a high water retention (Video S5). The reproducible volume changing meant that incredibly soft materials with a high liquid content are applicable to various biological and clinical research areas, from osteoporosis through tissue regeneration to hemorrhage control.

Figure 4d illustrates another kind of actuating method, where the hydrogel absorbed light energy and caused a local shape deformation, causing the spider-shaped hydrogel to activate, corresponding to the applied laser beam. In the light-fueled reconfiguration, the formed matrix absorbed photon energy and converted it into mechanical properties. The amplitude, location, frequency, and speed of the shape-changing properties passively depended on the applied laser beam (seen in Video S6). Here, both the laser pressure [49] and osmotic pressure in the water [50] contributed to the local shape morphing. Without the osmotic pressure (we evaporated the water off), the responsive activity of the spider hydrogel decreased significantly (seen in Video S7).

Furthermore, we fabricated the tadpole-shaped hydrogel, which swung its tail shape using the swelling effect (seen in Video S8), where the in-plane tail bent in air but straighten in water (Figure 4e). As a typical reverse process, we could prove the shrinkage of the tail to the initial state (seen in Video S9). In the discussion, the critical factor, besides the material affecting the bending and stretching, was the groove depth in the tail, which has previously been explored as a mechanism for shape deformation using a self-folding theory based on Timoshenko's theory [51].

Then, we fabricated a smart two-layer structure (Figure 5a), and the swelling happened out-plane in a perpendicular direction. The planar hydrogel bent upward reversely, and a part of the hydrogel relocated on the substrate due to intrinsic adhesion. The trick for the reconfigurable two-layer structure was the uneven scanning space of two layers. Therefore, the densities of two layer (seen in the SEM image of Figure 5a) varied a lot, leading to an uneven swelling or shrinkage degree and inducing shape morphing on the upper layer. The interface between the two layers was linked by smooth covalent bonding, with no mismatch of the traditional dual-layer design for actuation. Notably, the bending direction was perpendicular to the substrate (Figure 5b), implying a direction control using a twolayer structure. No fracture or physical damage was found in any of the shape-morphing hygromorphic hydrogels. Reverse programmability also meant that the functional groups were well maintained during and after TPP. The micro-structured hydrogels required only several seconds for shape reconfiguration, outperforming those bulky hydrogels of slow diffusive swelling rates [52,53] due to the micro/nanoscale surface effect, which made them more applicable for various aqueous environments.

#### *3.3. Heat-Induced Shrinkage Behavior*

Besides the humidity or light stimuli for responsiveness, the heating process also led to a self-bending action similar to an artificial muscle (Figure 6). The unique features found by heating, provide possibilities for sensing or actuation as well. If heated, the water uptaken by hydrogel would evaporate. Subsequently, the created surface tension changed to form a shrinkage-based 3D structure. The interspacing of adjacent nanowires modulated the bending degree. Illustrated by the flower (Figure 6a), heart (Figure 6b), and grid structure (Figure 6c), heat-induced deformation became predictable and useful. The hydrogel detected the temperature shifting in the ambient environment and changed its surface tension in the macroscope view.

**Figure 5.** (**a**)The model of dual-layer design, its zoomed-in view, and a side-view SEM image of the differentiated two layers. The upper layer has a higher density of arranged nanowires, and the lower layer has a relatively smooth density; scale bar of the SEM image is 5 μm; (**b**) a two-layer design realizes upward bending out-plane, a group of four two-layer hydrogels demonstrates upward bending observed at height-changed focus. The scale bar of SEM image is 50 μm, scale bar of the CCD image is 100 μm. *3.3. Heat-Induced Shrinkage Behavior* **Figure 5.** (**a**)The model of dual-layer design, its zoomed-in view, and a side-view SEM image of the differentiated two layers. The upper layer has a higher density of arranged nanowires, and the lower layer has a relatively smooth density; scale bar of the SEM image is 5 µm; (**b**) a two-layer design realizes upward bending out-plane, a group of four two-layer hydrogels demonstrates upward bending observed at height-changed focus. The scale bar of SEM image is 50 µm, scale bar of the CCD image is 100 µm. *Micromachines* **2022**, *13*, x FOR PEER REVIEW 10 of 13

ing: (**a**) the closure of flower mimic hydrogel working as a thermal gripper, where the scale bar is 50 μm; (**b**) an unevenly shrunk hydrogel from initial symmetrical heart shape, where scale bar is 50 μm; (**c**) the model, SEM image, and shrinkage process of a grid-shaped hydrogel, the scale bar is 50 μm. **Figure 6.** TPP-fabricated thermal-responsive hydrogel for temperature-controlled shaping–morphing: (**a**) the closure of flower mimic hydrogel working as a thermal gripper, where the scale bar is 50 µm; (**b**) an unevenly shrunk hydrogel from initial symmetrical heart shape, where scale bar is 50 µm; (**c**) the model, SEM image, and shrinkage process of a grid-shaped hydrogel, the scale bar is 50 µm.

The heterostructure consisted of solidified hydrogel nanowires, and the smooth spacing resulted in a divergence in shrinkage behavior. This divergence caused residual stress at the molecular level and caused the inward-direction contractile surface tension to accu-

als, causing a disorderly shrinkage. Subsequently, we fabricated a symmetrical heart shape, where the heating process showed an asymmetrical shrinkage (Figure 6b). As seen in Figure 6c, another grid hydrogel self-folded into an out-plane uneven ball through heating. Conclusively, the uneven distribution of the geometry intensified the self-folding character and decreased the responsive time. The underlying mechanism for the controllable shape morphing could be found in the explanation section on surface tension (Supplementary Materials). The mechanics platform (seen in Figures S7 and S8, Supplementary Materials) further verified the tunable mechanical properties for reverse shape

In this study, we succeeded in developing a composite hydrogel material sensitive to a water/light/heat environment with a 532 nm femtosecond laser TPP. Compared to the

**Figure 6.** TPP-fabricated thermal-responsive hydrogel for temperature-controlled shaping–morph-

morphing.

**4. Conclusions**

The heterostructure consisted of solidified hydrogel nanowires, and the smooth spacing resulted in a divergence in shrinkage behavior. This divergence caused residual stress at the molecular level and caused the inward-direction contractile surface tension to accumulate. As seen in Figure 6a, the heat-transferring process differentiated in the eight petals, causing a disorderly shrinkage. Subsequently, we fabricated a symmetrical heart shape, where the heating process showed an asymmetrical shrinkage (Figure 6b). As seen in Figure 6c, another grid hydrogel self-folded into an out-plane uneven ball through heating. Conclusively, the uneven distribution of the geometry intensified the self-folding character and decreased the responsive time. The underlying mechanism for the controllable shape morphing could be found in the explanation section on surface tension (Supplementary Materials). The mechanics platform (seen in Figures S7 and S8, Supplementary Materials) further verified the tunable mechanical properties for reverse shape morphing.
