Recent Advances in Multi‐Photon 3D Laser Printing: Active Materials and Applications

Since the pioneering work of Kawata and colleagues in 1997, multi‐photon 3D laser printing, also known as direct laser writing, has made significant advancements in a wide range of fields. Moreover, the development and commercialization of photocurable inks for this technique have expanded rapidly. One of the current trends is the transition from static to active printable materials, often referred to as 4D microprinting, which enables a new degree of control in the printed systems. This review focuses on four primary application areas: microrobotics, optics and photonics, microfluidics, and life sciences, highlighting recent progress and the crucial role of active materials, including liquid crystalline elastomers, hydrogels, shape memory polymers, and composites, among others. It also addresses ongoing challenges and provides insights into the future prospects in the different fields.


Introduction
[4][5] This method, also known as direct laser writing, uses a tightly focused laser in the near-infrared (NIR) spectral regime to induce nonlinear multiphoton absorption processes, which leads to photopolymerization within the focal volume.By moving this focus through a suitable photocurable ink, complex 3D microarchitectures can be created at the micro-and nanoscale.Today, in contrast to initially exclusively custom-made laser setups, several companies offer commercial microfabrication devices expanding the range of often called 4D (micro)printing. [14]The additional dimension refers to the ability of printed materials to evolve with time, for example by changing shape or size.This feature can be achieved by employing active materials, often called smart materials.Key classes of smart materials employed in 4D (micro)printing comprise liquid crystalline elastomers (LCEs), [12] smart responsive hydrogels [10] as well as shape memory polymers (SMPs), [15] but in some cases also composites including inorganic fillers.LCEs are a unique class of materials that combine the properties of both liquid crystals and elastomers. [16,17]They are known for their ability to undergo reversible, changes in response to external stimuli and exhibit remarkable mechanical and optical properties, making them valuable for a wide range of applications, including soft robotics, and optical devices (see Sections 2 and 3).Their actuation relies on the anisotropic volumetric changes associated with the transformation of the initially ordered crystalline phase -in most cases a nematic phase -toward an unordered isotropic phase.A common approach to achieve LCE printable formulations is the use of suitable photopolymerizable mesogens with mono-or multi-functional photoreactive molecular moieties (=liquid crystalline monomers and crosslinkers) together with additives such as photoinitiator or solvent. [11]dditionally, controlling the mesogen alignment during 3D fabrication is crucial.
Hydrogels are hydrophilic polymeric networks with high water content, and therefore, soft materials (Young's moduli in the range of kPa). [18]Their ability to mimic natural tissues, biocompatibility, and their tunable physical and chemical properties make this material of great interest, especially for applications in the fields of life sciences (see Section 5).By using Figure 1.Overview of the main active materials and four core application areas of multi-photon 3D laser printing, which will be discussed in the presented review.Adapted with permission. [20]Copyright 2020, The Authors.Adapted with permission. [21]Copyright 2017, The Authors.Adapted with permission. [22]Copyright 2021, The Royal Society of Chemistry.Adapted with permission. [23]Copyright 2023, Wiley-VCH.Adapted with permission. [24]opyright 2020, The Authors.Adapted with permission. [25]Copyright 2023, Wiley-VCH.Adapted with permission. [26]Copyright 2019, American Association for the Advancement of Science.Adapted with permission. [27]Copyright 2018, The Royal Society of Chemistry.
functional hydrophilic monomers in the formulation, responses to various stimuli including temperature, pH, or solvent can be implemented into the printed material.Typical monomers are Nisopropylacrylamide (NIPAAm), acrylic acid (AAc), acryl amide (AAm), or modified natural proteins.
SMPs are a third class of smart materials with interesting properties in 4D (micro)printing.These materials can be programmed physically to arbitrary temporary shapes and maintain this form until the recovery of the initially fabricated shape is initiated by exposure to a suitable stimulus. [15]Importantly, the dynamic characteristics of this material class do not rely on alignment strategies as it is the case for LCE but are solely based on the network characteristics such as chain segment composition as well as crosslinking density.In the case of thermally induced SMPs, a temperature stimulus is employed to overcome a specific transition temperature -either an implemented melting temperature or the glass transition temperature of the polymeric network -inducing the geometrical change.Compared to responsive hydrogels and LCEs, SMPs exhibit superior mechanical properties with Young's moduli in the range of hundreds of MPa to GPa and applicability in a variety of media. [15,19]ombining the advantages of smart materials and the precise microfabrication offered by multi-photon 3D laser printing, new opportunities have been created in a wide range of application areas.Herein, we have identified four core application areasmicrorobotics, optics and photonics, microfluidics as well as life science -with concurrent research efforts in multi-photon 3D laser printing and active materials (Figure 1).In the following, we present a general overview of the remarkable progress of recent years achieved in each of the areas with a special focus on the role of printable materials.Furthermore, present and future challenges as well as the perspectives of the field will be discussed.

Microrobotics
[30][31] Following the ongoing trend of miniaturization in soft robotics, the search for new approaches toward the generation of functional microdevices has become of great importance.Combining efforts in material design as well as engineering high-performing active 3D microstructures using multi-photon 3D laser printing, a wide variety of robotic systems in the micro-and nano regime have been developed in the recent decade. [7,14]In this section, microactuators relying on the use of active printable materials will be first introduced.Next, recent mobile systems including microswimmers and microrobots equipped with diverse functionalities will be presented.Furthermore, control strategies for locomotion along with various recent systems and their application in drug delivery, cell delivery, and microobject manipulation will be discussed.Last, a selection of more complex micromachines composed of multiple components or multiple materials will be given.

3D Microactuators
To achieve complex microrobotic devices, the design of active structures (=microactuators) is the first key step.As stated above, smart polymeric materials used for this purpose include LCE, responsive hydrogels, and SMPs.By a proper design, these materials offer a wide space of responsive features upon external stimuli including heat, [32,33] light, [34,35] or pH changes. [36,37][40][41] Furthermore, 3D microactuators made of this material class exhibit the advantage of reversible actuation extending their range of applicability drastically.A key step to ensure actuation is the defined and precise molecular alignment during fabrication.Standard methods employed for such alignment utilize sandwich cells transferring homogenous vertical, horizontal, or splayed director alignment from modified substrates to the encapsulated LCE ink.For example, planar alignment is achieved by glass substrates coated with thin polyimide (PI) or polyvinyl alcohol (PVA) layers that are rubbed with a cotton cloth to create microgrooves on the surface. [42,43]Circumventing such coating protocols, Guo et al. presented a strategy allowing voxel-by-voxel encoding of nematic alignment in fabricated 3D microactuators by multi-photon 3D laser printing the microgroove structure directly on the glass substrate using commercial IP-S ink (Nanoscribe GmbH). [44]Successive 3D printing allows the generation of microactuators with defined director alignment as demonstrated by their anisotropic solvent-induced shrinkage behavior.In more recent work, the same working group presented an approach allowing the generation of arbitrary 3D director fields. [45]This was achieved by printing individual "voxels" -cubes with aligned director fields and assembling them to the final actuator structure by UV gluing.Although surface transfer methods are established and widely used, a frequently faced problem is the limited height transfer of the alignment information -restricting the achievable microstructure size in general.Recently, Münchinger et al. demonstrated such full 3D control over the alignment of the director by applying a quasi-static electric field which was controlled during the polymerization process. [46]Employing a custom-designed cell including electrodes, the authors were able to print dynamic 4D microstructures with arbitrary spatial distribution of the director in three dimensions.The authors were able to adjust the alignment for each voxel individually by dynamically changing the applied electric field (see Figure 2A).Along with LCE microactuators that respond to temperature changes [47] , the incorporation of chromophores offers an attractive alternative for controlled actuation.First reported systems employing azobenzene as chromophore were demonstrated by the research group of Wiersma fabricating a microhand and a microwalker actuator. [38,40]Trying to circumvent the issues associated with the use of UV light, scientists worked toward the implementation of photoreponsive units allowing the use of longer wavelength light in the visible or NIR range offering biocompatibility and large penetration depths.For example, Chen et al. employed a printable nanocomposite composed of a suitable mesogenic monomer and crosslinker. [48]The addition of thiol functionalized gold nanorods to the ink allowed for reversible NIR-induced actuation in fabricated microactuators.Another strategy allowing facile access to multiresponsive microactuators that can be actuated at different wavelengths of light was demonstrated recently by our group. [34]By using five different dyes, the photoresponse was tunable in the entire visible spectral range (400 to 700 nm).Integration of dyes with orthogonal absorption wavelengths led to wavelength-selective actuation behavior.Combining this simple approach with the previously described electric field alignment strategy [46] allowed for the fabrication of complex optomechanical LCE-based metamaterial structures (see Figure 2B). [35]n addition to LCE-based microactuators, hydrogel-based materials have also been exploited for the fabrication of microactuators.For example, Hippler et al. exploited the thermoresponsive properties of poly-N-isopropylacrylamide (PNIPAAm) offering a lower critical solution temperature (LCST) at 32 °C. [32]Increasing the temperature above the LCST induces a phase transition of PNIPAAM from hydrophilic to hydrophobic properties, which is also accompanied by a visible shrinkage in printed microstruc-tures.Lowering the temperature below the LCST reverses the phase transition opening the path toward multiple actuations.Using the same system, Spratte et al. were able to show an efficiency increase of the thermoresponsive actuation behavior in dependence on geometry as well as the adjustability of mechanical properties by fabrication parameters. [49]This approach, termed grey tone lithography, gave access to a range of different materials featuring distinct mechanical, thermal, and dynamic properties -utilizing only one ink system.In another example, Decroly et al. fabricated structural elements for bending compression and twisting deformation, demonstrating complex geometries based on hydrogels featuring advanced kinematics. [50]Deng et al. presented photoresponsive PNIPAAm-based microactuators by printing a carbon nanotube doped bilayer structure composed of a responsive layer featuring assembled metamaterial unit cells and a solid nonresponsive layer both printed using similar fabrication conditions. [51]By smart exploitation of the proximity effect -usually reducing resolution quality -the inner part of the metamaterial unit cells was crosslinked with low density allowing for example the fabrication of an artificial light-driven beating heart.Besides using carbon nanotubes, other photothermal agents such as gold nanorods were employed for photoresponsive shape deformation too. [52]H is another stimulus that has also been of great interest for responsive 3D microactuators.For instance, Jin et al. demonstrated pH-responsive microstructures with an ink system consisting of NIPAAm, AAc, a water-soluble photoinitiator, and a multifunctional crosslinker to promote printability. [36]he addition of polyvinylpyrrolidine, an additive often used in connection with AAc, adjusted the ink viscosity for multiphoton 3D laser printing.As shown by Zandrini et al., a higher ink viscosity extends the range of applicable energy doses without the formation of bubbles or structural defects. [53]he pH-induced size change originates from pendant carboxylic groups featuring electrostatic repulsion of present carboxylate anions under basic conditions. [36]Similarly, Scarpa et al. reported a pH-responsive ink based on the copolymerization of 2carboxyethyl acrylate with poly(ethylene glycol) diacrylate (PEG-DA) crosslinker components. [54]Huang et al. demonstrated a modular design strategy toward complex microstructures including a printed race car that can be transformed upon a suitable pH stimulus to a humanoid robot (see Figure 2). [37]Chen et al. presented micromachine component architectures featuring the ability to self-lock and unlock upon exposure to suitable pH [55,56] and Zhang et al. generated microcrab structures mimicking arthropod's actuation characteristics upon stimulation with suitable pH conditions. [57]Importantly, the inclusion of structural color features on the microrobot platform allowed additional potential for tracking.
Although extensively investigated at the macroscale, the use of SMPs as microactuators started very recently.Zhang et al. employed commercially available ink VeroClear (Stratasys Inc.) in combination with an elastomeric matrix, allowing multi-photon 3D laser printing of stimuli-responsive color filters with transition temperatures ≈40 °C. [58]Elliot et al. focused on a synthetic approach by preparing methacrylate functionalized monomers featuring suitable chemical functionalities for possible postfunctionalization after printing. [59]A first approach to bridge the gap between the well-known world of macroscale printed SMPs Figure 2. A) In situ alignment method allowing full control about LCE director alignment during the microprinting process.Adapted with permission. [46]opyright 2022, The Authors.B) LCE-based optomechanical metamaterial structures generated using in situ director alignment via electric field and strategy and stimuli incorporation by a facile dye absorption approach.Adapted with permission. [35]Copyright 2022, The Authors.C) A microprinted pH-responsive hydrogel race car transforming into a humanoid structure in response to a suitable stimulus.Adapted with permission. [37]Copyright 2020, The Authors.D) Thermally induced shape memory cycle of microprinted SMP-based actuator.Adapted with permission. [33]Copyright 2022, The Authors.
and multi-photon laser printing of SMPs was presented by our group.We reported a shape memory polymer ink system allowing successful printing at the macroscale via digital light processing, a standard vat photopolymerization method, as well as on the microscale via multi-photon 3D laser printing. [33]This was achieved by using an ink system composed of exclusively commercially available components including isobornyl acrylate as chain builder and a dual system of stiff and soft crosslinkers allowing advantageous printability characteristics as well as remarkable shape memory effect for both size regimes (see Figure 2D).Recently, Jeske et al. employed thiol-ene chemistry to generate polymeric networks of lower crosslinking density exploiting a step growth mechanism during microprinting. [60]In very recent work, Minnick et al. prepared sensor-like actuators employing the shape memory effect of integrated IP-Visio (Nanoscribe GmbH) microfibers for force measurement in a liquid medium. [61]

Design Parameters
An inherent feature of active microswimmers and mobile microrobots is their ability to perform locomotion upon stimulus in a controlled predetermined way.Thus, these systems must possess geometries and propulsion strategies allowing efficient, remotely controlled locomotion in the desired media.Changing an object from daily encountered dimensions (cm to m regime) to the size of micrometers has dramatic consequences on the propulsion strategy, that is, the relative importance of different physical processes at different size regimes must be considered.For example, microrobots acting in a fluid medium operate under conditions of low Reynolds (RE) numbers.As demonstrated by Purcell introducing the Scallop theorem, efficient locomotion at the microscale is only possible by the use of nonreciprocal locomotion strategies. [62]Microorganisms that face similar environmental conditions have developed for example screw-like motions such as in rotating helical flagella of bacteria (Escherichia coli, E. coli) or flexible beating flagella of spermatozoa.These systems have served as a source of inspiration to solve the challenges of artificial microswimmers.Other modes of nonreciprocal motion were also applied by exploiting surface frictional forces in spherical microrollers and microwalkers.
Having optimized the shape of a microrobot according to such design schemes, the next step to decide is the source of propulsion.Especially in view of biomedical applications ideal control of the microrobots occurs remotely and with suitable high precision utilizing a biocompatible and noninvasive method.In recent years, major scientific focus was located on the exploitation of different stimuli including magnetic fields, acoustic waves, light, piezoelectric stimulation, or chemical reactions resembling the concept of active Janus particles.
Magnetic fields especially show advantageous features as locomotion stimulus in terms of noninvasiveness and biocompatibility.To implement magnetic responsiveness in printed structures, two main strategies are employed by using either a postfunctionalization or a responsive ink approach.In the first approach, microswimmer structures are printed in the desired 3D shape and subsequently rendered magnetically responsive by surface functionalization with thin layers of magnetic material.Usually, these coatings are introduced by physical vapor deposition of magnetic metals and alloys based on nickel or cobalt. [63]ecently, this procedure has been expanded toward magnetic alloy CoNiP wet metallization to reduce shadowing effects and increase cost efficiency. [64]To increase the biocompatibility, an additional layer of titanium is often added afterward.Post functionalization was also successfully achieved by surface decoration with magnetic nanoparticles. [65]In the second approach, a composite photocurable ink including magnetic nanoparticles is employed for direct multi-photon 3D laser printing of magnetic microstructures.Following this strategy, the implementation of superparamagnetic iron oxide nanoparticles (SPIONs) has proven to be a successful route toward biocompatible magnetic microstructures.Recently, the use of ferromagnetic iron platinum nanoparticles was proposed to replace SPIONs to increase the magnetization and locomotion efficiency and more precise actuation. [66]nother type of locomotion stimulus that rise in popularity is acoustic fields.For utilizing this stimulus, the microrobot is typically printed in the form of a bullet or spheroid designed with a hollow room inside the shape for the capture of an air bubble.This air bubble translates the oscillations of the acoustic field stimulus applied toward motion.In contrast to conventional swimming motion via corkscrew locomotion or rolling motions of magnetically actuated systems, acoustically stimulated locomotion happens via a unidirectional surface slipping mechanism.Implementation of magnetic control by suitable coating allowed improved directionality of the acoustically induced locomotion by applied magnetic fields. [67,68]Acoustic fields appear to be biocompatible rendering this locomotion technique in principle excellent for biomedical treatments.
Recently propulsion via chemical reaction is exploited as a locomotion stimulus.In most cases printed initially nonresponsive microrobots are selectively coated with catalytic platinum or nickel, allowing the generation of a stream of oxygen bubbles in the presence of hydrogen peroxide.[71] For example, Xin et al. combined the catalytic activity of platinum nanoparticles with magnetic actuation via ferroferric oxide nanoparticles and pH-responsive hydrogels [72] to fabricate fish-shaped microswimmers able to perform magnetic, catalytic, and hybrid propulsion to move within channel systems and overcome obstacles.
A further locomotion strategy uses light as an active stimulus as shown by Li et al. in 2020. [73]After multi-photon 3D laser printing microrockets with a commercial ink SU-8 and selectively depositing gold nanoparticles on their propulsion parts, controlled photothermal heating of the microrockets' rear part was achieved which resulted in a temperature increase of the fluidic environment behind the robot and a net forward motion.By multiplying the amount of such micronozzles, the achievable speed can be tuned toward higher values making this system promising for motion inside complex fluids such as blood in the arterial vessel system.

Applications of Mobile Microswimmers and Microrobots
Target Drug Delivery: One major application field for microrobots is targeted drug delivery allowing minimally invasive therapies enabling more efficiency due to less drug dose and thus fewer side effects.Mobile microrobots that can be loaded with drugs and precisely controlled through the human body appear as optimal solutions for this purpose as they are able to easily pass narrow blood vessels and human organoids due to their small dimensions.However, several obstacles must be overcome to reach a fully functional system.The fabricated microrobots must be biocompatible as well as biodegradable and must not produce an immune response.Cabanach et al. reported such nonimmunogenic stealth magnetic microrobots that appear invisible to the immune system. [74]The authors employed methacrylate functionalized zwitterions carboxybetaine and sulfobetaine for helical microswimmer fabrication.Remarkably, various immune cells showed no aggressive behavior toward the biocompatible and non-toxic microswimmers even after the exhaustive inspection in vitro.Furthermore, the microrobot offers additional potential due to possible drug loading by encapsulation or surface loading.Ceylan et al. showed a 3D biodegradable microswimmer allowing theranostic cargo delivery and release tasks. [75]Combining gelatin methacryloyl (GelMA) with SPIONs a double-helical microswimmer was fabricated allowing complete degradation to nontoxic products via matrix metalloproteinase-2 (MMP-2) enzyme.Cargo released was implemented in the initial steps of the biodegradation process by MMP-2-induced swelling.The remaining SPIONs were antibody tagged allowing cancer cell labelling.Using a similar material, Chen et al. produced biodegradable microrobots featuring targeted DNA transport abilities for therapeutic purposes. [76]Besides encapsulation of DNA functionalized nanoparticles, the GelMA-based spherical microrobots offered by cationic surface functionalization loading of DNAcargo too.Another approach toward biocompatible microswimmers was presented by Wang et al. in 2019 by fabricating microswimmers based on metal-organic frameworks (MOFs). [77]he initial helical microswimmer body was printed using commercial resin IP-L (Nanoscribe GmbH) and functionalized with biocompatible and pH-responsive ZIF-8 crystals -the MOF component -on the surface enabling pH-responsive drug release as well as magnetic actuation.In a follow-up work the system was rendered biodegradable by the use of GelMA as an ink component. [78]n order to further reduce possible safety risks due to a lack of biocompatibility, Ceylan et al. proposed magnetically responsive drug transport systems based on blood-derived biomaterials by printing helical microswimmers and -rollers. [79]Controlled cargo release was demonstrated by the exploitation of the pH-controlled reversible shape memory effect.Moreover, material components allow enzymatic degradability with proteinases reducing risks for long-term toxicity.Envisioning a system allowing the removal of all potentially harmful components after microrobot drug delivery, Lee et al. fabricated a PEG-based helical microrobot encapsulating an anticancer model drug during multi-photon 3D laser printing. [80]After approaching the location of action surface connected magnetic nanoparticles were divisible by NIR irradiation in the presence of reducing conditions imitating the cancer cell environment, offering removal via a magnetic field as demonstrated in vitro.
After arrival at the aimed position, many microswimmers exploit passive diffusion as a mechanism to deliver the therapeutic toward its destination.A representative example of this is the magnetic needle-type robot presented by Lee et al. allowing attachment on target microtissue thus offering stable drug delivery. [81]Targeting cancer therapy Park et al. developed a drug delivery system showing potential for hyperthermia therapy. [82]mplementing iron oxide nanoparticles in the microrobots' PEGgel matrix not only yields stimulus-responsive locomotion but also permits heating of the microrobot surrounding environment to temperatures above 40 °C -allowing for example cancer cells to suffer irreversible damage -by conversion of electromagnetic energy to heat.Besides normal diffusion release mode fabricated swimmers allow actively controlled drug release overcoming slow diffusion dynamics in this way.
Introducing additional stimuli explicitly for drug release is often connected with an increase in complexity in material design, however, allows the preparation of more efficient drug delivery microtools.Typical stimuli initiating drug release are light, [74,83] pH changes, [77,84] or enzymes typically present at the delivery site. [75]Due to the high level of spatial control light appears favorable as a drug release stimulus, whereby the trend goes from UV light toward NIR light, due to better biocompatibility and skin penetration depth.Employing NIR light as a stimulus for drug transfer Song et al. reported a remarkable microrobot system inspired by puffballs allowing the transport and liberation of large drug loads (see Figure 3A). [83]Employing a multistep fabri-cation protocol, hollow spheres featuring a top hole were printed using commercial IP-S ink, followed by magnetic coating, and centrifugation-based drug loading finalized by sealing the hole with a polycaprolactone cap containing photothermal dye IR780.Thus, cap removal by melting via NIR irradiation allows controlled drug delivery.Another spheroidal multiresponsive system was shown by Lee et al. fabricating a pollen grain-inspired microrobot. [84]By performing a three-step multi-photon 3D laser printing protocol, multimaterial structures consisting of a pHresponsive inner sphere, a magnetically actuatable crust featuring pollen grain-inspired spikes, and an outer thermoresponsive crust layer were fabricated.Upon temperature response, the spikes can be exposed on demand offering surface anchoring on tissue.The pH-responsive inner shell acts as a drug carrier and controlled release switch.However, efficient drug release was only possible at pH 11 without notable cargo liberation at a physiological pH of 7.4 due to lack of swelling.
Cell Transport: Along with drug delivery, another core application envisioned for microrobots is the precise delivery of cells: for example, for stem cell transplantation or neural cells to induce neural growth and repair biological damage.Many microswimmers employed for this purpose feature either cubic geometries with structured surfaces or spherical geometries.Scaffold-type structures are often employed to achieve improved cell attachment and larger surface area to increase the cell loading capacity of individual microswimmers.These geometry types also offer the advantage of physical protection of loaded cells.For example, magnetic spherical porous microtransporters were fabricated by Li et al. allowing cell transport to target position with successful cell release demonstrated in vivo. [88]In a follow-up work, Wei et al. presented a degradable cell transporter of similar design (see Figure 3B). [85]Degradability was this time implemented by a change of the ink material from SU-8 to PEG-pentaerythritol triacrylate (PETA) copolymer composited with magnetite nanoparticles instead of prior used magnetic coatings.
Jeon et al. fabricated a variety of magnetic scaffold geometries for stem cell delivery. [26]The tested geometries included cylindrical and hexahedral scaffolds as well as spherical and helical geometries.Transported neural stem cells successfully proliferated and differentiated.Moving toward more controlled systems, Yasa et al. designed a microtransporter with a recapitulated stem cell niche allowing programmable and active cell delivery. [89]By employing a two-step protocol, the cylindrical base structure was first printed followed by pattering the inside hollow cylinder with functionalized collagen, hyaluronan, and fibronectin mimicking a stem cell niche.Successful preservation of stemness was achieved as well as controlled differentiation inside the stem niche by suitable stimulation.Envisioning a robotic device for neural cell delivery enabling selective connection of neural networks for biomedical investigations, Kim et al. prepared a magnetically actuatable microrobot featuring a microgrooved platform (see Figure 3). [86]The specific microgroove design allowed controlled guidance of neurite outgrowth for selective reconstruction of neural networks in vitro.
Another area within the application field of cell transport lies in reproductive medicine.Transport of fertilized early-stage embryos -termed zygotes -toward the fallopian tube appears as a critical step during artificial fertilization due to the complexity of the process and the low rate of successful artificially induced  [83] Copyright 2022, The Authors.B) SEM picture of unloaded and loaded spherical cell transporter featuring scaffold architecture.Adapted with permission. [85]Copyright 2020, Wiley-VCH.C) SEM image of magnetically actuatable microgrooved platform for controlled guidance of neurite outgrowth and selective reconstruction of neural networks.Adapted with permission. [86]Copyright 2020, The Authors.D) Sperm transport system targeting in vivo assisted fertilization.Adapted with permission. [87]Copyright 2022, The Authors.
pregnancies.In order to overcome this challenge, Schwarz et al. developed a strategy for a microswimmer-assisted zygote intrafallopian transfer, allowing zygote capture, transport, and release at the destined location. [20]icromanipulation: Another promising area for microswimmers lies in the field of micromanipulation of biological entities as well as nonliving objects.Possible applications can be envisioned in areas like microsurgery or lab-on-chip devices com-prising uses as smart sensors or intelligent microfluidic channels respectively active components.In many cases, microobject manipulation occurs via direct physical contact. [63,90]Apart from that, microswimmer systems employing in addition noncontact manipulation strategies via the generation of hydrodynamic vortices were developed, offering more flexibility toward the requirements of the cargo. [91]Using acoustic and magnetic actuation Mohanty et al. presented a microrobot mimicking the fluid expulsion mechanism of cephalopods. [92]By acoustic power modulation, the microrobot can grasp nearby objects and release them at destined locations.The authors envisioned applications as microsieves or for direct cell manipulation.Baker et al. demonstrated a different option for different swimming behaviors by platinum-coated torus-shaped microswimmers combining catalytic propulsion and magnetic direction control. [93]The swimmers were able to perform tasks of high complexity including scavenging and sort of passive and charged microspheres as well as cargo release on demand.
An often-used approach to manipulate respectively relocate microparticles or cells includes optical trapping of them and manipulation by optical tweezers.However, many types of particles are not manipulatable using these conventional methods and biological entities can be damaged by the tightly focused laser.By combining optical and hydrodynamic trapping concepts, Būtait ė et al. circumvented this problem elegantly by designing a swimming rotor microsystem that can be optically actuated achieving precise object manipulation without irradiating the respective entity directly. [94]herapeutic nanoparticles have shown major difficulties in terms of transport.One hurdle to overcome is the limited transport efficiency from the blood vessel toward the targeted tissue.In order to overcome this challenge, Schuerle et al. developed two strategies allowing the mass transport of nanoparticles using printed helical microswimmers as well as magnetic bacteria. [95]cting as micropropellers the printed structures can steer the nanoparticles via convective flow toward the vessel wall, allowing them to accumulate and penetrate adjacent tissue.
Micromanipulators allowing targeted microsurgeries inside body tissue are another important future application field of microswimmers.Envisioning a system toward precisely targeted obstructive interventions, Lee et al. created multiresponsive magnetic microswimmers featuring orthogonal response in terms of size change by either temperature change, pH change, or Ca 2+ ions. [96]ithin the last few years, the concept of biohybrid systems has risen.Instead of simple manipulation of biological cells, a more symbiotic approach was realized employing the cell as a propulsion source perfectly adapted to microscale conditions.A highly interesting system based on spermatozoa was extensively studied by the group of Schmidt to employ these biohybrid systems for drug delivery, artificial fertilization, as well as cancer therapy.By combining spermatozoa as propulsion force with printed magnetically guiding parts, efficient locomotion and control behavior was achieved.Using this approach systems for drug transport strategies for hydrophilic [97] and hydrophobic [98] therapeutics, artificial fertilization, [87,99,100] and cancer therapy [97] were established as well as locomotion strategies in more sophisticated environments such as blood vessels. [101]Exemplary Rajabasadi et al. reported multifunctional sperm microcarriers that were responsive to multiple stimuli envisioning the goal of in vivo assisted fertilization (see Figure 3D). [87]Remarkably, advanced strategies for drug transport increased by capturing multiple loaded sperms within microflakes for efficient motion was reported too. [102]Based on their perfect adaption to conditions at the microscale as well as good biocompatibility these approaches can possess a promising future.

Toward Complex Micromachines and Microrobots
The use of micromachines able to perform more intricate tasks often requires higher levels of complexity in the microrobotic design and architecture.One step toward this challenge is the implementation of several materials either by multistep printing or inclusion by postprinting processing steps, decoupling in this way active from passive structural units.For example, Hu et al. created magnetic micromachines composed of active Janus microparticles which are linked together to actuator geometries. [103]Combining gold-coated Janus particles, with rigid and soft printed links and structural elements allowed the fabrication of a variety of micromachine configurations including a lizard-like walking robot or tetrahedron arrays.By utilizing a NIPAAm-based ink system, Xin et al. fabricated a humanoid-like robot in the first laser printing step and functionalized it on the joints via photoreduction with photothermal silver particles for light-controlled actuation (see Figure 4A). [104]nspired by nature, Ma et al. combined the advantages of hard-bodied and soft-bodied parts to a complex smart gripper able to catch and release microobjects (see Figure 4). [105] hard nonresponsive 3D skeleton was fabricated in the first printing step using commercial ink SU-8 followed by the addition of smart pH-responsive artificial muscle elements using a bovine serum albumin (BSA) ink system.In a remarkable work by Jia et al.ATP-triggered complex microrobots were achieved.The authors fabricated shape-morphing robotic structures from printed protein-based modular units.[106] These were coated with a motorized actomyosin exoskeleton allowing shape deformation through contractility achieved by myosin molecular motors translating chemical energy toward mechanical work as demonstrated for a robotic hand.By use of photocaged ATP modules, local control over actuation was demonstrated utilizing a light stimulus (see Figure 4C).[106] One way to achieve complexity in geometry and function very fast is the design of reconfigurable systems allowing assembly of different structural units having distinct functionality.Moving a step toward this direction, Alapan et al. developed a mobile compound micromachine system offering reconfigurable and hierarchical assembly.[107] By smart use of shape-encoded interactions by taking advantage of dielectophoretic forces between laser-printed static micromachine units and mobile magnetic and self-propelled actuator structures the authors achieved different active micromachine geometries including a mobile microcar or a microrotor structure as well as controlled assembly of individual structural units.
Another way to achieve functionality in complex micromachines is the use of suitable geometry and the design of active elements for later actuation.Kaynak et al. prepared for example acoustically actuatable micropump devices based on a PEG-DA and PETA ink. [108]In detail, compound micromachines were composed of a microchamber featuring sieves for particle selection by size and attached micropump devices allowing size selective filtering.In another work, Smith et al. fabricated for example microhydraulic systems taking inspiration from the geometry of spiders. [109]igure 4. A) Humanoid microrobot with photoresponsive features allowing large spatial control in actuation.Adapted with permission. [104]Copyright 2023, The Authors.B) Microgripper based on a combination of hard nonresponsive parts and active elements.Adapted with permission. [105]Copyright 2020, The Authors).C) Light controllable actuation of a printed microhand by use of photocages ATP modules.Adapted with permission. [106]opyright 2022, The Authors).

Optics and Photonics
Since its emergence, multi-photon 3D laser printing has played a key role as a core manufacturing technique for microdevices, especially in optics and photonics.[117][118][119] Furthermore, the interest in the incorporation of stimuliresponsive features to enable additional control in microdevices has grown in recent years.[129] The Main smart material class used for these applications are LCEs allowing advantageous remote control by a suitable temperature or light-based stimulus. [130]For example, Sandford O'Neill et al. demonstrated microprinted DOE that can be switched on and off by use of electric voltage. [120]In the following, we will highlight several recent examples of emerging applications where active materials have played a key role:

Smart Color Filters
In nature, color appears in two forms: chemical color and physical color.In the case of physical color -often termed structural color -the origin lies in the refraction, diffuse reflection, diffraction, and interferences of periodic micro-and nanostructures.[133] These embedded pigments in chemical color-based microstructures can undergo bleaching by gradual oxidation and lead to a performance loss in terms of discoloration over time.The optical stability of structural colors is directly associated with the structural integrity of the micro-and nanostructures and thus with material stability.The recent implementation of stimuli-responsive features into structural color devices has enabled new applications in areas of anti-counterfeiting labels or smart color filters.Recently, Dong et al. reported multi-photon 3D laser-printed structural colors based on the commercially available photoresist SZ2080 that shows different expansion ratios in dependence on the surrounding medium, accessing in this way the possibility toward encryption and decryption devices by simple medium exchange. [134]By changing from ethanol to air and subsequently aqueous medium decryption of a Taichi diagram structure was successfully demonstrated.Instead of complete immersion in the solvent, Delaney et al. reported photonic arrays showing reversible structural color response to vapors of different solvents including isopropanol, ethanol, and water under atmospheric conditions. [22]Exploiting the swelling property of the polymerized hydrogel network resulting from the acrylamide monomer and PETA, a reversible optical response was achieved due to the polarity change of the surrounding vapors rendering this photonic device ideal for sensor applications.Moving to pH-responsive hydrogels, Chu et al. reported a new system suitable for use in neutral biomedical conditions. [135]The pH-responsive features were achieved using AAc, whose carboxylic acid functionalities exhibit strong electrostatic repulsion in acid or basic solutions.The 3D-printed microstructures were able to cover almost the entire red-greenblue color space of the CIE 1931 chromaticity diagram.Demonstrating the applicability, an amphichromatic fish structure was fabricated by printing woodpile structures and reversible discoloration changing the pH stimulus in the physiological range below and above pH 7 (see Figure 5).Using shape memory polymers, the working group of Yang was able to produce structural color filters allowing remarkable thermoresponsive control. [58,136]oreover, it was shown that cholesteric LCEs offer responsive color changes to controlled stimuli exposure due to their intrinsic anisotropy. [137]Ritacco et al. further showed that the color of the printed structures based on cholesteric LCEs can be tuned in situ by the printing parameters. [138]In another example, del Pozo et al. presented the fabrication of microstructures which changed their structural colors as well as shape in response to temperature and humidity. [139]

Imaging Systems and Microanalytical Devices
One important application of micro-optical devices is imaging in medical therapy.Remarkable examples are reported by Li et al. presenting systems for optical endoscopy (see Figure 5B). [140,141]esembling biological eyes, the generation of artificial microcompound eyes was recently intensified due to their large potential as micro-optical imaging systems. [21,142,143]These lenses are inspired by insect compound eyes and the first approaches for implementation on the microscale have been considered attention in the last years since they could offer a wider field of view compared to classical lens systems.A combination of this geometry type with active materials was recently performed to achieve the first steps toward a tunable miniature imaging system allowing dynamic control in focal length.In detail, Ma et al. printed a micro-compound eye lens (μ-CE) using BSA, a protein-based pH-responsive biomaterial (see Figure 5C). [144]Upon changing the pH of the solution, the authors were able to tune the focal length and the field of view significantly due to size change upon swelling and shrinkage.Furthermore, combining the BSA μ-CE structure with a microprinted spherical base of a commercial non-responsive material yielded ≈400% of focal length tuning at a fixed FOV.Remarkably, the change in focal length of the presented system was highly reversible in 100 cycles without showing fatigue.
Apart from imaging devices further complex systems envisioning analytical characterization were generated using multiphoton laser printing.One remarkable example is a scanning probe microscope fabricated by the group of Koos (see Figure 5D). [145]The author employed microprinting for the fabrication of the scanning probe microscope engine.In addition, optical elements of the device including microlenses or -mirrors featuring the necessary optical quality were fabricated in accordance with a microprinting methodology employed in a former work. [146]A spectroscopic microdevice was reported by Toulouse et al. fabricating a Adapted with permission. [135]Copyright 2022, Wiley-VCH.B) Digital microscope image of the lens-in-lens directly printed on a no-core fiber targeting a microendoscopic device.Adapted with permission. [140]Copyright 2022, The Authors.C) Left: Working principle of the pH-responsive protein-based compound eye.Right: Exemplary SEM image of a microprinted compound eye.Adapted with permission. [144]Copyright 2019, Wiley-VCH.D) SEM image of the scanning probe microscope engine.Shielding structures were used to prevent undesired metal coating of lens surfaces.Adapted with permission. [145]Copyright 2019, The Authors.miniature spectrometer for the visible range by a smart combination of multiphoton laser printing with inkjet technology as the main fabrication technique. [147]

Microfluidics
Microfluidic devices became an enormous toolbox for research on the fundamental behavior of liquid transport as well as for many applications ranging from separation and sensing to labon-a-chip and organ-on-a-chip technologies.Various applications already proved their tremendous impact on daily life by becoming inevitable for disease screenings such as for the coronavirus disease 19 (COVID-19), human immunodeficiency virus, and different types of hepatitis. [148]Research on microfluidics started in the late 1980s and developed rapidly in the 1990s.The rise was further facilitated by new advanced manufacturing technologies like soft lithography.Today, most microfluidic components such as connectors, switches, and valves are commercially available and can be connected for the construction of complex devices.This assembly requires multiple steps making the fabrication tedious.Therefore, recent developments in manufacturing focus on the use of additive manufacturing.][151][152][153][154] Compared to photolithography, this method allows for easily tailorable 3D master structures with the possibility to employ curved surfaces in the z-dimension while being a cost-efficient alternative to electron beam lithography.Although multi-photon 3D laser printing is also a promising tool for the direct fabrication of micro-sized structures for microfluidic devices, its role in the fabrication of microfluidic devices is still in its infancy.Strikingly, multi-photon 3D laser printing offers not only the potential to facilitate existing protocols but also enables possibilities in terms of achievable 3D structures and applied materials in these channels which cannot be obtained by Figure 6.A) SEM image of an entirely 3D printed microfluidic device for the fabrication of a fillable drug carrier.Adapted with permission. [23]Copyright 2023, The Authors.B) SEM cross-section image of the drug carrier for the filling process.Adapted with permission. [23]Copyright 2023, The Authors.C) SEM images of a bistable pneumatic actuator prepared by multi-photon 3D laser printing on a glass capillary (bottom) in its two different states.Applying different pressures inside the glass capillary opened or closed the gripper.Adapted with permission. [163]Copyright 2023, The Authors.D) SEM image of a spinneret which was printed inside of a microfluidic channel structure.Adapted with permission. [27]Copyright 2018, The Royal Society of Chemistry).E) Concept (left) and optical microscopy images (right) of printed pH-responsive actuators inside a microfluidic channel for trapping particles and yeast cells.Changes in pH allowed for reversible swelling of the AAc-containing hydrogel.Adapted with permission. [164]Copyright 2019, The Royal Society of Chemistry.F) Schematic setup for multi-photon 3D laser printing with a microfluidic chamber for fast fabrication of fluorescent multimaterial structures.All steps were performed without removing the structures out of the setup.Adapted with permission. [165]Copyright 2019, The Authors.
any other means.In the following, different approaches will be described in detail including recent examples in literature.Note: Recently, 3D microprinted channel structures have been gaining attention for biological and medical applications (e.g., in dynamic cell cultures or tissue models).These studies combining microfluidics and life sciences will be not discussed here but in the next section.

3D Printed Microfluidic Devices
Multi-photon 3D laser printing has been successfully employed as a versatile tool for the fabrication of entire microfluidic devices.For example, in 2022, Sun et al. reported a procedure to print microcarriers that were filled through a microfluidic channel (see Figure 6A,B). [23]Filling of the carrier solution was possible after carefully designing the channels to control the surface tension, pressure, material waste, and connection between the system and the filling nozzle.After filling, the carriers were closed with a stimuli-responsive polycaprolactone sealing that released the encapsulated solution upon heating or NIR irradiation.Since these printed carriers transport liquids instead of traditional hydrogelloaded beads, they might carry higher loading of for example drugs and at the same time offer an instantaneous release.
Even though printing larger microfluidic devices entirely offers the flexibility of accessible structures, the fabrication speed of multiple microfluidic devices is limited since every 3D printing step requires large amounts of printing time.This subject was addressed by Van der Velden et al.In 2020, the authors reported the use of a commercially available positive photoresist AZ 4562 (Microchemicals GmbH) which gives rise to the entire microfluidic device minimizing the printing time by printing only the channels with their approach. [155][158][159][160] The field of printing parts on microfluidic channels was recently expanded by printed components actively interacting with the surroundings.In this approach, microfluidic channels were, for example, coupled with imaging or served as delivery systems.Kramer et al. for example fabricated a microfluidic atomic force microscope cantilever tip which was successfully used for imaging, cell puncturing, and precise fluid manipulation. [161]The fabrication consisted of two separate printing procedures.First, a macroscopic fluidic channel was printed with stereolithography.The tip was printed using multi-photon 3D laser printing in a second step to achieve microscale resolution.An example of a biomedical application was reported by Mu et al. in 2023 who printed a microfluidic channel and integrated it into a photonic neural probe. [162]With this device, the authors were able to inject and release caged fluorescein in a controlled way.
One way of combining microfluidics with active components was achieved by multi-photon 3D laser printing of components which changed their structure upon pneumatic pressure inside of the channel.Barbot et al. presented a procedure toward printed micropistons inside of a glass capillary tip. [166]The piston moved upon applied gas pressure inside the capillary.Depending on the sealing structure of the capillary, this piston was further used for force sensing or gripping applications.Power et al. expanded this system in 2023 toward grippers which showed bistable behavior (see Figure 6C). [163]By oxygen plasma etching, the authors achieved 400 nm thin substructures in their chevron-style bistable gripper design able to undergo structural changes toward pressure changes.Alsharhan et al. combined printed pneumatically actuated grippers with printed transistor structures. [167]In their work, two microprinted transistor structures were successfully coupled to two printed grippers in a COP microfluidic device.The two grippers on the surface of the microfluidic channel responded toward pneumatic pressure separately giving access to multiple actuation patterns.The transistor structures were printed directly inside the microfluidic device.
Moving further toward multi-photon 3D laser printing inside of microfluidic channels, one of the first examples was reported by Lölsberg et al. in 2018 with the fabrication of a spinneret inside of and on a microfluidic PDMS channel (see Figure 6D). [27]This spinneret allowed for wet spinning of individual aligned polyacrylonitrile fibers with a diameter of 2.7 μm.In 2019, Oellers et al. prepared a fluidic micromixer by printing in a predefined channel. [168]][173][174] It is noteworthy that multi-photon 3D laser printing was so far mainly used for printing solid barriers without the possibility of actively changing their shape.In 2019, Hu et al. demonstrated a microfluidic device that contained stimuli-responsive structures able to change their shape upon stimulus. [164]The authors prepared hollow ring-like structures for reversible trapping and releasing of particles and cells depending on the pH environment (see Figure 6E). [164]The core of the trapping mechanism was a microprinted hydrogel consisting of AAc, NIPAAm, and dipentaerythritol pentaacrylate (DPEPA).Upon increasing the pH above 9, the acidic residues deprotonated, and charging-induced swelling occurred.Here, the swelling process of printed microrings was accomplished within 200 ms.Decreasing the pH of the solution inside of the microfluidic chamber below 9 recovered the printed hydrogel in its original shape.The authors were able to trap and release polystyrene particles and yeast cells of selected size by choosing the pH and distance between printed hydrogel columns.Moreover, microprinting of stimuli-responsive structures in microfluidic channels can also be used for sensing different solvent conditions. [175]n alternative to printing directly inside of the microfluidic channel is to assemble the microfluidic channel onto the previously printed structure.This approach was successfully employed by Nouri-Goushki et al. in 2019 for the study of cell behavior on submicron patterned 3D structures. [176]

Printing with Microfluidic Devices
Multi-photon 3D laser printing in combination with microfluidic devices has also been explored, for example, for its benefits in the preparation of multi-material structures. [165,177]In a pioneering work, Mayer et al. built a setup consisting of a microfluidic chamber integrated directly into the 3D printer (see Figure 6F). [165]ere, the microfluidic device serves as an excellent platform for the sequential pumping of different resins or washing solvents through the channels.This approach ensures fast ink exchange and quick overall fabrication times.By using this approach, the authors demonstrated the possibility of a complex 3D microstructure for safe features using seven different materials, including dyes and solvents.
Another interesting aspect of employing microfluidic devices in combination with multi-photon 3D laser printing is the possibility to use a continuous flow during printing.[180][181] For example, Lölsberg et al. prepared a microfluidic device that was successfully used for the 3D printing of a polymeric microtube in a vertical fluid-flow setup. [178]icrofluidic channels for dynamic cell cultures with multiphoton 3D laser printing fabricated micrometer-sized features have gained a lot of attention recently for offering increased perfusion of liquids. [182,183]Biological and medical applications of microprinted channel structures for developing various tissue models have been a matter of recent research.Current studies combining microfluidics and life sciences are discussed in the next section on life sciences. [25,182,184,185]

Life Sciences
Precise 3D printing of biocompatible materials is one key to further progress in the field of life sciences.In contrast to other techniques such as extrusion or filament printing, multi-photon 3D laser printing offers a unique tool by giving access to 3D structures with sub-micrometer resolution and high speeds.For this purpose, various photocurable inks offering biocompatible materials have been developed in the past -some of which have become commercially available.[188] A general trend has become more and more pronounced in the last five years: the focus of recent works shifted from using photocurable inks leading to highly crosslinked materials toward new biocompatible materials with interesting mechanical or functional properties.][191][192][193] The growing interest in these materials can also be seen by the commercialization of new biocompatible inks offering elastomeric materials.
In the following sections, we aim to provide an overview of multi-photon 3D laser printing for life science applications with a focus on future perspectives of using active materials and forming higher-order cellular structures.First, we present recent work on single-cell scaffolds focusing mainly on structures with active materials.Subsequently, the possibilities of multi-photon 3D laser printing for organoid scaffolds, tissue engineering as well as the possibilities to directly print in living organisms are discussed.
Figure 7. A) Scheme of multimaterial microscaffold for mechanical stimulation of cells.Swelling of the hydrogel upon the addition of free adamantanecarboxylic acid (AdCA) leads to the bending of the TPETA and PETA substructures and to the stretching of the cell in the scaffold.Adapted with permission. [24]Copyright 2020, The Authors.B) Confocal microscopy images of a printed gelatin-based dome that surrounded a cancer cell spheroid.The encapsulated cancer cells proliferated and escaped the dome after two to four days (not shown).Adapted with permission. [212]Copyright 2023, The Authors.C) Printed grid with embedded vessel substructures for effective dynamic 3D tissue culture (left the image of the printed structure, middle close image of the printed structure, right a scheme of vessel perfusion and aggregated stem cells after seeding on printed structure).Adapted with permission. [182]Copyright 2023, The Authors.D) SEM image of a microchannel cell scaffold printed with commercially available IP-DIP (Nanoscribe GmbH) ink for developing neuronal tissue models.Adapted with permission. [213][213] Copyright 2020, American Chemical Society.E) SEM image of parallel multi-photon 3D laser printing printed microporous tubes in a microfluidic chip mimicking microcapillaries in the blood-brain barrier.Adapted with permission. [25]Copyright 2017, Wiley-VCH.

Cell Scaffolds
To date, one of the most exploited applications of multi-photon 3D laser printing in life sciences is the preparation of scaffolds for cells. [9]The advantages of multi-photon 3D laser printing in the preparation of cell scaffolds rely on the possibility to altering the printed structures in the micrometer regime.This tool offers a direct structural impact on single-cell behavior, perfusion properties, and cell arrangement.In general, two different procedures for studying cell behavior in 3D microprinted scaffolds are accessible.In the first and more common procedure, multi-photon 3D laser printing of scaffolds is performed in the absence of cells allowing the scaffold fabrication with cytotoxic inks.After development, the biocompatible scaffold is excessively washed with solvent to remove residual ink and photoinitiator prior to cell seeding.Expanding this procedure to multi-photon 3D laser printing of structures in the presence of cells was achieved by encapsulating cells in a biocompatible physical gel which can be photopatterned or photocrosslinked.[196][197][198][199][200][201][202] An extensively ongoing field of study deals with the interactions of microprinted materials with single cells focusing on the behavior of cells in confined scaffolds or on various substrates.][205][206][207] For a detailed overview and analysis of these effects on single cells, we kindly refer to a recent review on this topic by Link et al. [208] Recently, the investigation of single cells in dynamic environments has become possible by employing printable materials, whose properties can be changed on demand upon external stimuli.One of the first examples of such a material was presented by Hippler et al. in 2020 (see Figure 7B). [24]The authors created multimaterial microscaffolds that could stretch cells in a controlled manner.The responsive hydrogel, which led to swelling inside of these microscaffolds, was composed of acrylamide, acrylamide-functionalized -cyclodextrin (CD-AAm), and acrylamide-functionalized adamantane (Ad-AAm).Addition of adamantanecarboxylic acid (AdCA) in a postprinting step induced swelling of the hydrogel due to the competitive host-guest dynamics of the -cyclodextrin-adamantane system.The bioorthogonality of this stimulus allowed for the mechanical stimulation of cells without influencing inner cellular behavior.Zhang et al. have used prepared pH-responsive stimuliresponsive hydrogel structures for the preparation of dynamic cell scaffolds. [209]The hydrogel consisted of printed AAc as pH responsive monomer and PETA as a crosslinker.Upon increasing the pH from 6 to 8, the authors observed a significant expansion of the hydrogel which was reversible in over 40 cycles.By utilizing dynamic multi-foci processing, the authors created multiple structures in parallel and henceforth succeeded in speeding up the fabrication process.
Besides shape morphing, biocompatible materials with other functionalities have been reported.For example, Qin et al. reported a hydrogel consisting of polyvinyl alcohol hydrogel and an enzyme-degradable peptide crosslinker which was loaded with cells and 3D photopatterned with a dicysteine cell responsive RGD peptide using multi-photon 3D laser printing. [198]By controlled 3D photopatterning and removing the excess peptide, the functionalized hydrogel scaffold directed cell migration along the photopatterning regions.During the cell migration, the peptide crosslinker is degraded opening the path for the cell inside the gel.In another study, Gräfe et al. presented an enzymedegradable biocompatible material in 2020. [210]The photocurable ink consisted of poly(ethylene glycol) dithiol and a synthesized crosslinker which degraded in the presence of the enzyme chymotrypsin.Exposing the structures toward a solution of 1 mg mL −1 chymotrypsin for four hours fully decomposed the material.Lemma et al. were able to achieve selective positioning of different cells on microprinted scaffolds. [211]After multi-photon 3D laser printing, a photocurable ink consisting of an acrylated photoreactive molecule (photoenol, PE) with PETA or trimethylolpropane ethoxylate triacrylate (TPETA), maleimidefunctionalized single-stranded DNA (ss-DNA) reacted with the photoenol moiety under UV light.The result of the UV-induced Diels-Alder photoaddition was a ss-DNA functionalized surface which was later utilized for anchoring.Fluorophores and cells functionalized with complementary ss-DNA were selectively attached to the surface of the ss-DNA functionalized printed structure by DNA hybridization.By printing and functionalizing the surface with different ss-DNA, selective positioning of different fluorophores or cells was enabled.In an alternative approach, the surface functionalization was accomplished with a UV laser beam giving spatial control over the functionalization area.For this purpose, Lemma et al. used biotinylated maleimide which reacted with the photoenol moieties in the illuminated volume.Subsequent treatment of the surface with streptavidin allowed for selective binding of biotinylated ss-DNA to the remaining streptavidin binding pockets.

Organoid Scaffolds
Interest in organoid research has evolved tremendously in the last decade since it provides a simplified in vitro model for understanding tissue development and diseases. [214]The development of organoids in 3D microprinted precise micrometersized 3D scaffolds has been recently explored mainly in biocompatible cell-embedded hydrogels.Precise 3D printing around organoids allows for controlling biological processes, such as size, shape, cell identity, migration, and morphogenesis during their development.In 2020, Urciulo et al. showed for example that embedding human small intestinal organoids in photosensitive gelatin and multi-photon 3D laser printing a wall around the organoid induces a cue that gives rise to columnar epithelium. [215]In this work, 7-hydroxycoumarin-3-carboxylic acid (HCC) functionalized gelatin was utilized together with a multiarm HCC-poly(ethylene glycol) conjugate to embed the organoids.The photocurable ink was printed without the addition of an external photoinitiator by (2 + 2) photoaddition of the HCC groups.In a follow-up study in 2023, the authors presented a detailed study of organoid development in a confined space of the same material presenting data on organotypic spinal cord, lung cancer organoids, liver organoids, and organotypic culture of lung epithelium rudiments. [216]In this work, Urciulo et al. used a basement membrane extracellular matrix for encapsulation of the organoids and added the previously described HCCfunctionalized photoresist on top of the encapsulation gel. [216]After incubation, the authors were able to print solid structures inside of the gel.Subsequent removal of the basement membrane extracellular matrix led to the printed HCC-gelatin structure with encapsulated organoids.This multi-photon 3D laser printing by a hydrogel-in-hydrogel approach allowed for live imaging of organlike 3D cultures with very high resolution in hydrogels with tunable mechanical properties at desired points in time.
Encapsulation of cancer micro-spheroids was also possible with a commercially available gelatin-based photocurable ink (see Figure 7B). [212]Taale et al. embedded cancer cell spheroids and proved successful in printing dome-like structures on top of the cells or only on parts of them. [212]After removing unpolymerized ink, the authors analyzed the escape of cancer cells out of the spheroid behavior.This method demonstrates an easily accessible procedure to study 3D tumor models.Very recently, Guillame et al. presented the spontaneous assembly of a cell spheroid of human adipose-derived stem cells inside a printed hydrogel bucky ball. [191]Further, the authors demonstrated the spontaneous assembly of stem cells in various shapes such as tetrahedron and cubic structures depending on the printed matrix and proved their differentiation potential.This method offers an interesting way to control the shape and dimension of the cell assembly.
Organoid scaffolds have also been combined with microfluidics.In 2023, Grebenyuk et al. for example demonstrated a large-scale perfused tissue with printed vessels in the dimensions down to 10 μm (see Figure 7C). [182]Strikingly, the authors ensured a permeability for water-soluble molecules like fluorescein within the 3D cell culture in less than 10 min.The hydrophilic printed scaffold for dynamic cell culture was prepared by combining hydrophilic, but cell-repellent PEG-DA with cell-adhesive PETA.Importantly, the printed structure did not significantly swell after development.The vessel-3D printed scaffold was further used in biological studies with aggregated human pluripotent stem cells to prove the concept of enhanced tissue health by increased nutrient supply and transport of metabolites.By studying tissue health, the authors showed a fivefold increase in cell proliferation and reduction of cell apoptosis throughout the course of differentiation.Furthermore, employing the printed vessel structure in dynamic cell culture showed increased differentiation times and allowed for culturing of cells over the course of at least two months.

Tissue Modeling
In addition to single-cell and organoid scaffolds, multi-photon 3D laser printing of scaffolds for tissue engineering and modeling has been explored in recent years.The design and structuring of model tissues with multi-photon 3D laser printing is advantageously facilitated by implementing micropores in the subcellular dimension giving access to highly perfusive scaffolds.Moreover, these porous 3D tissue scaffolds can be generated with high spatial control over pore locations, sizes, and distribution.So far, various artificial tissue models have been prepared and studied such as bone, [217] cartilage, [218] cardiac, [25,184,185,194,205,219] neuronal, [185,213,[220][221][222][223] retinal, [224] and liver lobule [225] tissue.Their development increases not only the understanding of fundamental processes.If combined with microfluidic devices, it could further offer a promising platform for potential organ-ona-chip applications. [25,182,184,185]In particular, recent work on the most studied models, namely neuronal and vascular tissue, is presented.

Neuronal Tissue
Multi-photon 3D laser printing of structures for neural tissue engineering gained considerable attention in the last years by printing rods or channel-like scaffolds that offer directed control over neuron alignment.This model allows for example to study the development of neurite outgrowths and length, size of continuous networks, and branching behavior of neurons.Fendler et al. presented a proof of concept for 2.5D single-cell neuronal circuits inside of a printed microchannel arrangement. [222]The authors prepared a tower-like array using a commercial photoresist which was connected via microchannels.Biocompatibility of the microprinted structure was ensured by atomic layer deposition of passivating aluminum oxide.Before seeding murine cerebellar granule cells, an additional coating step with poly-D-lysine was performed to allow cell adhesion to the structures.The neurites grew in the printed microchannels as intended.The neurons in these 2.5D arrangements showed similar characteristic action potentials as vital neurons in 2D cell culture indicating proper neuronal function.Following up on this study, Harberts et al. prepared similar 3D microscaffolds and seeded them with mature neurons derived from human pluripotent stem cells (see Figure 7D). [213]he neurites grew inside the 3D-printed microchannels between the printed towers and connected to a network.The neurons showed spontaneous excitatory postsynaptic currents and fire action potentials across the neuronal network as vital neurons.The authors proved that it was possible to translate the previous findings in 3D scaffolds and that this system possesses the potential to be used in 3D brain-on-a-chip applications.In addition to the microchannel approach, printed grids showed the alignment of neurons along the grids forming 3D networks. [220]In 2022, Barin et al. studied epidermal growth factor receptors in patient-derived glioma cell cultures after their successful colonization on microprinted scaffolds. [226]This model was also used by Akolawala et al. to investigate proton-induced DNA damage in glioblastoma populated on microscaffolds. [227]Future improvements of the model would include the implementation of different materials to reduce the stiffness of the used material (≈1 GPa).

Vascular Tissue
In addition to neuronal models, (neuro)vascular models were developed and refined in the last few years.For example, Marino et al. prepared a 3D real-scale model structure of the blood-brain barrier in 2018. [25]This microprinted tubular structure was the size of 10 μm and mimicked the structure of brain microvessels (see Figure 7E). [25]The authors co-cultured endothelial and glioblastoma cells on the structures and modeled the flow parameters inside the porous capillaries.Furthermore, the diffusion of dextran out of the microtubes was investigated in a microfluidic chip with and without cells.Permeability and fluid flows resembled in vivo physiological parameters.Hence, this structure offers an excellent in vitro platform for investigations on the permeability of anticancer agents.Expanding this porous microcapillary system toward a dual channel system was performed by Buchroithner et al. in 2021. [185]In this work, a model for a blood-brain barrier was proposed which offered the analysis of the permeability of nanoparticles horizontally across co-cultured endothelial and glioblastoma cells between two vertical flows.The system was created by microprinting a grid between the two flow channels which was covered with the cells upon seeding.The authors showed that some particles permeated across the membrane while others remained at the cell surface.Dobos et al. expanded the material scope toward thiol-ene photoclick gelatin hydrogels which allowed direct encapsulation of cells inside of the photocurable ink. [194]By embedding human umbilical vein endothelial cell spheroids in the ink and printing vascular structures in the range of arterioles (30 μm), venules (20 μm), and capillaries (10 μm) around it, the authors demonstrated controlled cell proliferation and migration along these structures.

Printing in Living Organisms
Multi-photon 3D laser printing has also been demonstrated to be compatible with living organisms.In 2020, Urciulo et al. were the first to print the previously described biocompatible HCC-PEG and HCC-gelatin photocurable ink in living mice. [215]For this purpose, the photocurable ink was injected with a syringe in the skin, brain, or muscle tissue of an anesthetized mouse and microprinted without harming the surrounding tissue.A second work covering multi-photon 3D laser printing of electronically conductive material in and on living tissue was reported by Baldock et al. in 2023. [228]A biocompatible non-toxic photocurable ink was introduced on and in an anesthetized transparent nematode worm (Caenorhabditis elegans) prior to printing.Printing inside the worm's stomach was possible after feeding it with a mixture of ink and bacterial paste (E.coli, OP50).These examples suggest that 3D printing inside living organisms could become a tool to achieve control over cell organization in living tissue for biological and physiological research purposes or in minimally invasive surgical techniques.

Summary and Outlook
The design of new functional and active materials for multiphoton 3D laser printing has rapidly increased in recent years resulting in a rich variety of promising applications.The main reasons are the combined advantages of multi-photon laser printing as a 3D microfabrication technique offering freedom in the 3D geometries and high resolution down to the nanometer regime, along with the dynamic features introduced using smart materials.Remarkable advances have especially been shown in the research areas of microrobotics, optics and photonics, microfluidics, and life sciences, where the development of new materials has led to tremendous progress from microdevice fabrication toward application.In the field of microrobotics, several promising systems for applications in drug delivery, cell transport as well as micromanipulation have been demonstrated.In the other key application fields, optics and photonics, multi-photon 3D laser printing has been established as the preferred fabrication tool for the new generation of active optical and photonic elements including smart structural color devices and advanced responsive imaging systems.Moving toward microfluidics, multi-photon 3D laser printing offers an attractive way to produce precise 3D structured channels and structures with the option to employ active responsive materials in these microfluidic to be used in a wide range of applications Furthermore, this approach has proven to be an impactful tool in the field of life science, for the precise 3D fabrication of responsive scaffolds for single cells, organoids, and tissue modeling.Additionally, 3D laser printing in living organisms has just become a reality with great potential for future applications in biology and medicine.
Despite the significant progress within the last years, there are still challenges to overcome to reach the next level.On the one hand, technical aspects such as faster printing in terms of volumetric output, higher precision as well as the high costs of laser printers are still an issue.[231][232][233][234] Furthermore, volumetric output increase was demonstrated by employing multi-foci approaches, giving access to parallelized micromanufacturing and thus drastically increased numbers of generated structures per time unit. [235]Such developments are of special value for the fabrication of optic or photonic devices as well as scaffolds for biological experiments, where the manufacturing of large numbers of samples is desired.
Along with progress in technical aspects, further research in the design of new functional materials suited for the different applications is required.Special attention should be paid to new biocompatible and biodegradable materials suitable for multi-photon laser printing, being the basic requirements for applicability in life sciences and proposed therapeutic strategies.Although several presented material examples show advantageous features, further studies determining the long-term toxicity and effects of degradation products are necessary.Especially, exchanging artificial exogenous components of the respective applications with a biological moiety as shown for the biohybrid approach can be a future pathway.To achieve this, the design of novel active biomaterials as well as coupling of microprinted elements with living microorganims must be targeted. [236]Furthermore, special focus must be placed toward more precision and complexity in the response of the printed devices in a specific environment, ranging from physiological conditions necessary for biomedical applications to confined spaces as in the case of microfluidics or optics.Also, new approaches toward advanced multi-stimuli responsive materials as well as modular assembly strategies are very promising.Such advanced control in the features requires well-defined properties of the printable mate-rial.To achieve this, structural control on the molecular level is essential. [237]o conclude, fascinating achievements have been made in the development of functional materials for multi-photon 3D laser printing.Despite the challenges outlined, active materials have tremendous potential and great progress is foreseen in all the application areas in the near future.

Figure 3 .
Figure 3. A) Left: Multistep-fabrication scheme of puffball-inspired microrobots; Right: SEM image of the exemplary unsealed and unloaded puffballlike microrobot.Adapted with permission.[83]Copyright 2022, The Authors.B) SEM picture of unloaded and loaded spherical cell transporter featuring scaffold architecture.Adapted with permission.[85]Copyright 2020, Wiley-VCH.C) SEM image of magnetically actuatable microgrooved platform for controlled guidance of neurite outgrowth and selective reconstruction of neural networks.Adapted with permission.[86]Copyright 2020, The Authors.D) Sperm transport system targeting in vivo assisted fertilization.Adapted with permission.[87]Copyright 2022, The Authors.

Figure 5 .
Figure 5. A) Amphichromatic fish structure composed of printed woodpile elements featuring reversible discoloration by change of the pH stimulus.Adapted with permission.[135]Copyright 2022, Wiley-VCH.B) Digital microscope image of the lens-in-lens directly printed on a no-core fiber targeting a microendoscopic device.Adapted with permission.[140]Copyright 2022, The Authors.C) Left: Working principle of the pH-responsive protein-based compound eye.Right: Exemplary SEM image of a microprinted compound eye.Adapted with permission.[144]Copyright 2019, Wiley-VCH.D) SEM image of the scanning probe microscope engine.Shielding structures were used to prevent undesired metal coating of lens surfaces.Adapted with permission.[145]Copyright 2019, The Authors.