Multi-material multi-photon 3D laser micro-and nanoprinting

Three-dimensional (3D) laser micro-and nanoprinting based upon multi-photon absorption has made its way from early scientific discovery to industrial manufacturing processes, e.g., for advanced microoptical components. However, so far, most realized 3D architectures are composed of only a single polymeric material. Here, we review 3D printing of multi-materials on the nano-and microscale. We start with material properties that have been realized, using multi-photon photoresists. Printed materials include bulk polymers, conductive polymers, metals, nanoporous polymers, silica glass, chalcogenide glasses, inorganic single crystals, natural polymers, stimuli-responsive materials, and polymer composites. Next, we review manual and automated processes achieving dissimilar material properties in a single 3D structure by sequentially photo-exposing multiple photoresists as 3D analogs of 2D multicolor printing. Instructive examples from biology, optics, mechanics, and electronics are discussed. An emerging approach – without counterpart in 2D graphical printing – prints 3D structures combining dissimilar material properties in one 3D structure by using only a single photoresist . A controlled stimulus applied during the 3D printing process defines and determines material properties on the voxel level. Change of laser power and/or wavelength, or application of quasi-static electric fields allow for the seamless manipulation of desired materials properties.


Introduction
Multiple materials in three-dimensional (3D) additive manufacturing, that is 3D printing, are analogous to colors in 2D graphical printing.A set of primary colors and "primary materials" are the base.The different primary colors or materials need to form inks, printable by a single instrument.Last, a small set of primary ingredients must create thousands of different colors (materials), without the need for physically/chemically mixing them within one 2D pixel or 3D voxel, respectively.In 2D graphical printing this step is accomplished by dithering 1 or half-tone printing.By filling, e.g., 50% of a white paper with black pixels, the observer perceives a homogeneous gray area, if pixel size and the spacing between the pixels are sufficiently small, i.e., unresolved for the observer.
Today's multi-material 3D material printing is less advanced than multi-color 2D graphical printing, which has long made its way into most people's homes.3D printing on the macroscale was presented by Swainson 2 and Hull 3 , almost 50 years ago.Swainson already realized the importance of nonlinearities.Using stereolithography, Kodama 4 realized early on 3D polymer structures.3D additive manufacturing on the micro-and nanoscale was first realized experimentally in the 1990s by Maruo and coworkers 5 .Herein, negative-tone photoresists were processed by a nonlinear optical multi-photon-based technique.
In this review, we focus on multi-photon approaches which have seen a rapid recent development.Multi-photon approaches include two-photon absorption as well as the use of more than two photons in the excitation process.Today, multi-photon approaches provide printing rates of up to about ten million voxels per second 6 .To illustrate the meaning of this speed, we mention in passing that commercial home office 2D inkjet printers operate at rates of nearly ten million pixels per second.The consumer perceives these speeds as sufficiently fast.Multi-photonbased 3D approaches structure matter with a resolution approaching sub-micrometer and nanometer feature sizes 7,8 .Such spatial resolution is crucial for many applications in photonics and electronics 9−12 and is inaccessible to most other 3D additive manufacturing approaches 6 .However, the vast majority of 3D printed objects and devices made along these lines has been composed of only a single polymeric material.Multi-material architectures are much less investigated than single-material architectures, yet, most real-life systems (microscopic and macroscopic, biological and artificial) contain a large number of different materials with vastly dissimilar optical, mechanical, thermal, and electronic properties.Just consider the ingredients of a modern mobile phone.Therefore, this review further focuses on multi-material multi-photon approaches.
The main challenge starts with the correct "primary materials " in analogy to the primary colors.For example, electrically conducting or semiconducting primary materials cannot be obtained by combination of any number of non-conductive polymers.Here we review existing materials that might serve as a working set of "primary materials".In the second step, we discuss processing dissimilar primary materials within 3D printed structures using a single machine tool.We divide the corresponding literature into two avenues.
In the first avenue, different photoresists -the counterparts of the colored inks -are combined to manufacture a targeted multi-material 3D structure.So far, this combination has been accomplished by intermediate manual processing steps, but automated multi-photon multi-material 3D printing systems are rapidly developing.
In the second avenue, a single photoresist delivers 3D printed material with different properties.There is no direct analogue in graphical 2D printing.The underlying idea is to impose a stimulus during the 3D printing process of each voxel, influencing the photo-reaction of the ink, such, that the emerging material properties can be varied locally and deterministically in 3D.Conceivable stimuli include the locally applied laser power (e.g., changing the polymer cross-linking density), a quasi-static electric field vector (e.g., aligning molecules with an electrical dipole), a magnetic-field vector (e.g., aligning magnetic dipoles), or the wavelength of the exciting light (e.g., initiating reactions of different components within a single fixed photoresist system).A single, highly optimized "ink", delivered to the printing zone (defined by the laser focus), creates highly resolved multi-material 3D objects.This concept can of course be expanded to multiple photoresists.This review presents the current state-of-the-art of multimaterial multi-photon 3D laser printing.
However, in this review, we will not cover the farreaching idea of 3D metamaterials or "meta-inks".Printing 3D metamaterials mimics dithering in 2D graphical printing.We refer the reader to recent review articles by us 7,13 and others 14,15 .Nature proceeds quite similarly.It achieves a vast variety of different effective material properties in animals and plants by architecting on a micrometer and nanometer scale by using only a limited number of building blocks, based on polysaccharides, proteins, and minerals 16 .Printing tailored 3D microstructures results in artificial composites, with effective optical, mechanical, thermal, and electronic properties that can be qualitatively dramatically distinct from those of the constituents.As for dithering in 2D, it is key that the characteristic feature sizes are sufficiently small such that the observer does not notice them and rather experiences an effective homogeneous continuum (analogous to a homogeneous color).

Multi-photon 3D laser printing
In multi-photon 3D laser printing (alternatively 3D laser lithography or 3D direct laser writing), one tightly focuses laser pulses into the volume of a photosensitive material, the photoresist.Multi-photon absorption restricts the excitation to the focal region only and avoids spillover effects common and unavoidable in one-photon absorption 17 .The shape of the focal volume -locus of the chemical reaction -can also be influenced by the light's polarization 18 .The nature of this chemical reaction can differ greatly.For example, in monomers, light induces a local cross-linking 19,20 .In metal salts, light induces a reduction leading to metal nanoparticles in solution 21 .By scanning the laser focus through the photoresist in three dimensions, the desired 3D architecture is defined, including the possibility of overhanging structures.We note that, unlike for other 3D additive manufacturing approaches, a supporting material is usually not needed to realize overhangs.For example, to 3D print a bridge, layerby-layer approaches need to print something underneath the bridge because material cannot be deposited onto air.After the printing process, the auxiliary material needs to be removed.Multi-photon 3D laser printing does not require such auxiliary materials as the polymerized part "swims" in the liquid monomer surrounding.Using galvanometric mirror scanners, focus speeds of up to about half a meter per second at sub-micrometer voxel sizes have been reported 6 .Scanning of the illumination with speeds approaching the vacuum velocity of light is possible by projection-based temporal focusing 22 .After writing is completed, a suitable solvent removes the unexposed parts.The chemistry and mechanism involved in the printing processes depends on the photoresist system 17,19,20 .However, even for the most investigated monomer-based photoresist systems, details of the reaction-diffusion kinetics in three dimensions are not well understood.The situation is yet less clear for metal salts as photoresists for 3D printing of noble metals.

Bulk polymers:
Commonly used photoresists are composed of a monomer, a cross-linker, a photoinitiator, a photoinhibitor, and a solvent.By tightly focused laser irradiation, the monomer rapidly and efficiently crosslinks into a bulk polymeric network 19,20 .Inks can be acrylate-based (e.g., IP series, Nanoscribe GmbH), epoxy-based (e.g., SU-8 series, MicroChem), organic-inorganic hybrids (OrmoComp, MicroResist Technology), or hydrogel-forming photoresists 23−27 .Many photoresists are commercially available and ready-to-use without complicated preparation, enabling rapid fabrication of mechanically and chemically stable structures.The mechanical, thermal, optical, and bio-compatible properties of these materials allow the design of unique functional structures for applications as cell scaffolds 28,29 , 3D metamaterials 30−33 , microoptical elements 34−36 , microrobots 37−39 , and functional surfaces 40−42 .Bulk photoresists can be mixed with inert polymers such as Poly(methyl methacrylate) (PMMA) to customize properties, leading for example to better mechanical strength and higher viscosity in the manufacturing of overhanging structures 43 .

Conductive polymers:
The efficient multi-photon 3D printing of conductive polymers is an open challenge.Due to the strong absorption in the near-infrared, many intrinsically conductive polymers which can be used in other 3D printing techniques are difficult to use in multi-photon polymerization processes 44 .Poly (3,4-ethylenedioxythiophene) (PEDOT) and its relatives are attractive for 1.7 × 10 3 Sm −1 electronic applications because of their high conductivity and stability.PEDOT also is biocompatible and allows its use in biological environments 45 .Multi-photon 3D laser printing by in situ oxidative polymerization of EDOT mixed in polyethylene glycol diacrylate 46 or by polymerization of an EDOT-dimer 47 were demonstrated.Another important conducting polymer is polypyrrole.Polypyrrole (Ppy) and its derivatives have been studied intensely in different fields such as sensing or batteries 48,49 .Ppy displays high conductivity, and environmental stability even in the presence of liquid electrolytes.Unfortunately, truly 3D laser printing of this polymer has not been realized yet.An important step into this direction was taken by Agarwal et al.They demonstrated the use of a femtosecond laser as a new method for the polymerization and patterning of Ppy.In particular, pyrrole-3-carboxylic acid (PCA), was used for the writing of conductive patterns within microchannels.Its higher water solubility leads to favorable adhesion to glass surfaces.A conductivity of has been measured from a PCA polymer pattern 50 .

Metals:
The high optical peak intensities occurring within the focal volume of multi-photon 3D printers also allow direct photo-reduction of metal salts into metallic micropatterns with nanoscale precision 21 .Here, the photoresist is usually composed of a metal salt, a photosensitizer, a reducing agent, and surfactants.All of these components are dissolved in water or in a polymer matrix.Silver 51,52 and gold salts 53,54 are popular, but noble metals such as platinum and palladium have also been reported 55,56 .The direct laser writing of metal patterns has led to promising applications in microelectronics 53 , sensing and detection 57 , microfluidics 58 , and split-ring resonators for optical metamaterials 59 .

Nanoporous polymers:
A porogen is a chemically inert component which is miscible with the photoresist in its unpolymerized state, but induces a phase separation after multi-photon exposure resulting in porous structures.Importantly, separated phases may not display a large refractive-index contrast.Otherwise, light is scattered during the printing process.During development, the porogen is washed out from the pores and a nanoporous and strongly light-scattering structure emerges 60 .Such nanoporous 3D printed architectures have potential applications in controlling diffuse light scattering, as nanoparticle filters in microfluidics, as super-hydrophobic surfaces, or as scaffolds for cell and tissue culture.

Glasses:
Many polymers have refractive indices in the range of and no polymer exceeds .While such values suffice for many applications in optics, e.g., for microlenses and photonic wire binds 61 , tight waveguide bends or 3D photonic-band-gap architectures require larger refractive-index contrasts.A number of inorganic materials, such as chalcogenide glasses, PbSe, and LiNbO 3 , has sufficiently large refractive indices and 3D structures have successfully been made via direct multi-photon laser printing 62−64 .An alternative is to mix inorganic nanoparticles into monomeric photoresists.Examples are silicon-based monomers mixed with zirconium acrylate and titanium acrylate 65,66 .In these approaches, the refractive index can be tailored by the composition.More recently, the Rapp group demonstrated two-photon 3D printing of fused silica glass 67 .Their photoresist contained 32.5 vol% silica nanopowder in a photocurable binder matrix consisting of 40 vol% hydroxyethyl methacrylate and 60 vol% of trimethylolpropane ethoxylate triacrylate as crosslinker.After the 3D printing process, the structures were sintered at temperatures up 1 300 °C to obtain pure silica glass.Related processes have recently also been published by other groups 68,69 .

Crystals:
Optically transparent micro-and nanostructures have also been accomplished by using Yttrium-Aluminium-Granat (YAG) crystals as a solid photoresist in the sense that the crystal has been exposed to light 70,71 .Following light exposure, selective etchants are used to produce voids within the YAG crystal.This ansatz is conceptually similar to that used in chalcogenide glasses (see previous section) 62 .

Natural polymers:
Many polymers discussed above and others have successfully been used for the preparation of extracellular bio-scaffolds to support and interact with living mammalian cells 72 .An interesting alternative are proteins as photoresists.By multi-photon polymerization of proteins, specific cell-affinity has been be realized 73 .Bovine serum albumin (BSA) 26 is crosslinked when Rose Bengal is used as photosensitizer.However, the mechanical properties of cross-linked proteins are inferior to those of polymers.In addition, they have to be kept in a buffer solution to maintain their shape.Gelatin, a widely available biopolymer, can overcome many of these issues 74 .Different groups have assembled 3D structures using gelatin and its derivates 75 .Besides BSA and gelatin, other proteins and biomacromolecules, have been used as well, including chitosan, avidin, biotin, and fibrinogen 76−78 .

Stimuli-responsive materials:
3D printable stimuli-responsive polymers, hydrogels, and organogels emerge as an important and growing class of materials that are only covered as stimuli-responsive ingredients here (Section of multi-material stimulusresponsive microrobotics), as the topic has been reviewed recently 79 .

Polymer composites:
Many polymers discussed above and others have successfully been used for the preparation of extracellular bio-scaffolds to support and interact with living mammalian cells 72 .An interesting alternative are proteins as photoresists.By multi-photon polymerization of proteins, specific cell-affinity has been be realized 73 .Bovine serum albumin (BSA) 26 is crosslinked when Rose Bengal is used as photosensitizer.However, the mechanical properties of cross-linked proteins are inferior to those of polymers.In addition, they have to be kept in a buffer solution to maintain their shape.Gelatin, a widely available biopolymer, can overcome many of these issues 74 .Different groups have assembled 3D structures using gelatin and its derivates 75 .Besides BSA and gelatin, other proteins and biomacromolecules, have been used as well, including chitosan, avidin, biotin, and fibrinogen 76−78 .

Multiple material properties from multiple photoresists
The sequential 3D printing of dissimilar photoresists -as described in the preceding section -appears as a simple and straightforward approach to fabricate multi-material 3D structures: After exposing and developing a first photoresist, a second photoresist is introduced and exposed, developed, etc.This procedure is illustrated in Fig. 1a.Herein, positioning accuracy is of utmost importance 86 .Yet, a couple of fundamental restrictions apply.For example, after 3D printing dense metal or metal containing parts, the architecture is no longer transparent to light.Furthermore, it scatters the incident light.Both aspects make subsequent controlled multi-photon exposures difficult.Even if all previously 3D printed components are optically transparent, caution has to be exerted.If, for example, the different components have dissimilar refractive indices, the resulting laser focus is prone to optical aberrations, leading to distortions of the position as well as the size of subsequently 3D printed voxels.The strength of these aberrations depends on the refractive-index contrast and on the thickness the light has to pass towards the focus.In principle, in analogy to optical microscopy, these optical phase aberrations can be precompensated by using a spatial phase modulator.This would require knowledge of the spatial refractive-index distribution or algorithms for compensation without such knowledge 63,87 .However, so far, such corrections have not been applied in the context of multi-material multi-photon 3D laser printing.In addition, good adhesion between the different printed materials is required for the integrity of the final 3D structure.Although this is usually not an issue when using polymeric photoresists due the presence of unreactive groups in the surface of the pre-printed structures which allow the formation of covalent bonds in the next printing step, it can be challenging when combining materials with very different properties and nature, e.g., metals and polymers or organic and inorganic materials.
Given these constraints, the following multi-material systems have been developed.Applications in biology, optics and photonics, responsive microrobotics, and microelectronics have been achieved.

Methods and systems for the assembly of multiple materials
One option is to perform the steps mentioned above and depicted in Fig. 1a manually 88−95 .Most published multimaterial 3D structures made this way are composed of merely two dissimilar photoresists and only few samples have been reported to be composed of more than 2 kinds of materials 92,96−98 .However, with increasing number of different photoresists, this approach tends to become terribly difficult and irreproducible because the previously printed structure must be realigned manually in each step.This has spurred the recent development of different automated systems capable of 3D printing multi-material architectures.
A first approach is illustrated in Fig. 1b.It is based on storing the different photoresists and solvents on a surface or plate that can be moved by an automated linear or a rotational stage 99−102 .To avoid contamination between the different materials, two cleaning tanks containing solvents are included.In addition, an air nozzle allows for cleaning and drying.Although one-photon absorption in the ultraviolet is used here, this approach could be translated to multi-photon polymerization.
The second approach is based on integrating microfluidics into the 3D laser printer.Sun's group attached a polydimethylsiloxane (PDMS) parapet onto a glass substrate to form a microfluidic chip for flexible 100 nm injection and emission of photoresists and developer.This on-chip multi-photon polymerization strategy allows insitu integration of multiple photoresists.Microrobots and actuators have been demonstrated by combining rigid and stimuli-responsive resists 103 .Lamont et al. introduced a vacuum system to perfuse the PDMS-glass microchannel with liquid-phase photocurable material or developer.Along these lines, a DNA-inspired microstructure with five different materials has been achieved 104 .The positioning errors for the different materials have been on the level.Mayer et al. integrated a reusable microfluidic chamber into a state-of-the-art commercial 3D laser printing system (cf.Fig. 1c).The chamber was connected to customized pressure-flow control units.Seven different liquids, including five photoresists and two solvents, were used.However, the approach can easily be scaled up to a larger number 105 .Microfluidic devices also allow to polymerize multiple materials simultaneously when multiple photoresists are injected at the same time 106,107 .

Multi-material systems for applications in biology
Tailored 3D scaffolds influence and direct proliferation, migration and maturation of different cell types 72 .To resolve more complicated biological questions, 3D scaffolds composed of multiple materials and different surface functionalities are required.Sequential processing gives scaffolds with distinct protein-binding and proteinrepellent properties (Fig. 2a).Selective cell adhesion sites on scaffolds provide control of cell adhesion and cell shape in three dimensions 89 .Specific functional sites can be realized by photoinduced selective grafting of maleimide and subsequent bioconjugation.In this method, a homogeneous topography without discernible topographical change can be maintained 108 .Based on the control of cell shape and growth with well-defined adhesion sites in 3D (Fig. 2b, c), quantitative image processing and mathematical modeling reveal that the fibroblast shape is not only determined by contractility, but also by elastic stress in the peripheral actin bundles 109 .With 3D scaffolds defining the shape of cells, external forces can be applied to specific adhesion sites of single cells, enabling quantitative statistical analysis 110 .As mentioned above, proteins are interesting in this context.However, it is difficult to stack extracellular matrix proteins, such as fibronectin, to fabricate 3D scaffolds extending over 100 μm.Sie et al. adopted a multi-material approach based on BSA in a first step.In a second step, human fibronectin was inserted at specific locations (Fig. 2d) 90 .Proteins can also be embedded into a photoresist microscaffold to increase the mechanical robustness of free-standing 3D protein microstructures 111,112 .Serien et al. demonstrated proteinaceous networks within one microscaffold 111 .By sequentially processing of different materials, 3D scaffolds with more than 2 constituents are possible (Fig. 2e, f).The combination of a protein-repellent photoresist with a protein-adhesive resist as well as a photoactivatable passivating resist has been demonstrated 91,92 .Selected domains of these structures can be addressed by two distinct proteins (Fig. 2f).
Incorporation of responsive materials into bio-scaffolds facilitate the active response of scaffolds to tailored 3D microenvironments 113 .In this study, protein-repellent trimethylolpropane ethoxylate triacrylate (TPETA) was used for walls; pentaerythritol triacrylate (PETA), which adheres proteins on its surface, as the beams, and most importantly, stimuli-responsive host-guest hydrogel as actuator cell stretching (Fig. 2g−i) -in essence a microstretch-bench for cells.

Multi-material systems for optics and photonics applications
2D fluorescent security features are ubiquitous, e.g., on Euro bank notes and certain passports.Going to three dimensions increases counterfeit security.Different colors corresponding to different photoresists are needed.An early demonstration was based on polymers doped with fluorescent semiconductor quantum dots 98 .3D laser printing provides more security against counterfeiting compared with 2D structures.The combination of multiple fluorescent materials allows complicated optical patterns to be embedded.Herein, a nonfluorescent photoresist (PETA) served as a cross-grid holder structure.Onto this holder, two different fluorescent photoresists containing semiconductor quantum dots with fluorescence emission wavelengths of 525 and 450 nm, respectively, were 3D printed.Finally, the 3D structure was encapsulated in PETA, resulting in a thin transparent polymer film with fluorescent markers in its interior.Early structures were manufactured manually (see above).Later 105 , the described microfluidic system described has been used for the 3D printing of security features containing 5 different ingredient materials (Fig. 3a).
Multiple materials are also needed for correcting chromatic aberrations of microoptical components.Using two different photoresists (IP-S and IP-Dip) with different refractive indices, an achromatic axicon and a Fraunhofer doublet were fabricated by the Giessen group 114 .The same group also demonstrated the integration of polarization control (IP-Dip used for high resolution) and focusing elements (IP-S used for low shrinkage) directly onto the end facet of an optical fiber (Fig. 3b) 115 .Such components can also be directly 3D printed onto the end facets of lightemitting diodes and onto optical chips.
By integrating stimuli-responsive materials, tunable optical systems become accessible.The Wiersma group designed, 3D printed, and characterized an integrated polymeric photonic circuit with waveguides, grating couplers, and ring resonators in a dense geometry 116,117 .The waveguides and grating couplers were made from rigid commercial IP-Dip photoresist (Nanoscribe GmbH), while the ring resonator was made from and actuated by a liquidcrystal elastomer.Light served as a stimulus to spectrally tune the response of the photonic circuit in a controlled and reversible manner (Fig. 3c).

Multi-material stimulus-responsive microrobotics
Responsivity is crucial for accomplishing tasks such as environmental remediation 118 , cargo transportation 82 , and therapeutics in confined spaces 84 .The most used stimuliresponsive materials in multi-photon 3D laser printing are hydrogels/organogels, which change their volume under stimuli, e.g., pH value 77 or temperature 119 .To achieve complex actuation, the fabrication of multi-material structures is required.Often, responsive and nonresponsive (passive) materials are combined.
A representative example is BSA-based hydrogel.At isoelectric pH, the BSA proteins are uncharged and assume their lowest volume.Once the pH value is changed, the protein chains become ionized because of deprotonation/protonation of the amino acid groups.The strong electrostatic repulsion between the BSA molecular chains leads to the swelling of the BSA structure.Sun's group developed a successive on-chip 3D laser printing strategy to distribute BSA and SU-8, which act as muscles and skeletons in 3D musculoskeletal systems 103 .The internal network of both BSA and SU-8 can be programmed at the microscale and the elasticity of the muscle as well as the stiffness of the skeleton can be tuned for flexible actuation.On this basis, a pH-responsive spider microrobot was realized (Fig. 4a, b).In addition, a crab claw-muscle system and a smart microgripper were fabricated, which allows for pH-controlled capturing and releasing of microtargets.All of these musculoskeletal structures exhibit fast response, good durability, and robustness after long-time storage.
Temperature is another important stimulus parameter.Poly(N-isopropylacrylamide) (pNIPAM) is a wellestablished polymer, exhibiting a substantial response to changes in temperature due its lower critical solution temperature.Employing pNIPAM, Hippler et al. designed micrometer-scale rigid tubes stimuli-responsive valves.The main body of the tubes was fabricated by PETA.A pNIPAM-based torus was written inside the tube as a valve.By changing the temperature between 45°C and 20°C, the cross section of the tube was changed by a factor of 2.5 119 .pNIPAM can also act as a supporting platform for the successive writing of IP-L photoresist.In this way, freely suspended microstructures of a second material like a skeleton can be inscribed into a temperature-responsive pNIPAM matrix 120 .Nishiyama et al. deposited Ag nanoparticles inside temperature-responsive pNIPAM microgels.By their plasmonic absorption resonance, the Ag nanoparticles efficiently convert light into heat, leading to reversible relative microgel volume changes as large as 86% 121 .
Another mechanism is solvent-induced swelling of a hydrogel in a network with rigid materials.Duan's group presented a solvent-responsive hydrogel, which consisted of acrylamide as monomer and the sodium salt of 2acrylamido-2-methylpropane sulfonic acid as the ionic comonomer for the 3D printing of a micropump.This micropump system comprised a microfluid channel out of a passive photoresist (SCR 500) and a responsive hydrogel film as valve.By alternating the solvent composition (ethanol/water), the hydrogel film bent and led to reversible absorption and discharge of the fluid 122 .Another hydrogel prepared from butyl methacrylate as a monomer and propoxylated trimethylolpropane triacrylate as a crosslinker was used for the fabrication of a slipping-block microdevice 123 .Sun's group combined a humidityresponsive polyethylen glycol diacrylate (PEG-DA) hydrogel and inert methacrylate-based material using a two-steps fabrication method.A directionally bending cantilever was accomplished based on a hydrogel part swelling or shrinking in response to humidity changes 25 .Liquid-crystal elastomers (LCEs) represent another class of stimuli-responsive materials that are multi-photon printable 124 .For an introduction to the working principle and composition of LCEs, we refer the reader to ref. 125.Note that, in contrast to ordinary polymers, LCEs exhibit a preferred axis commonly referred to as the director.Raising the LCE temperature from below to above their phase-transition temperature, LCEs perform a phase transition from anisotropic medium to an isotropic medium.Various 3D movements, such as bending, contraction, torsion, jumping, and rotation of LCE can be reversibly controlled by programming the LCE director.These movements are triggered by various stimuli such as temperature, light, pH value, and electric or magnetic fields 126,127 .Wiersma and co-workers have reported a series of multi-material microactuators made by multi-photon 3D printing of LCEs and ordinary photoresists.A first example is a microscopic walker, which is able to move on different substrates in a dry environment 128 .Its main body was made from LCEs, which act as muscles (see Fig. 4c), and the four conical legs were made using IP-Dip photoresist.More recently, a hominid inspired microhand composed of four LCE fingers and four IP-Dip nails was realized 129 .Two of the fingers have different molecular alignment compared to the other two and bend in different directions under optical illumination.This microhand grabs and holds small objects when illuminated by green laser light without specific requirements for the environment (see Fig. 4d).

Multi-material 3D structures for microelectronics
For microelectronics, electrically conductive structures are crucial.As discussed above, various metals have been printed via multi-photon induced reduction of metal salts.However, it has so far proven difficult to obtain complex 3D structures along these lines [51][52][53]130,131 .
In a first approach, photoresists with different functional groups are printed sequentially.Thereafter, the entire structure is selectively coated with metal layers due to the difference in functional groups.Using this approach, the Kawata group reported the fabrication of 3D silver/polymer conjugated microstructures 88 .Herein, photopolymerizable photoresists with and without an amide group were independently prepared.Using electroless plating, silver was selectively deposited on the surface of the amide-containing polymer parts (Fig. 5a).
The Fourkas group demonstrated the selective chemical functionalization of 3D polymer microstructures composed of an acrylic polymer and a methacrylic polymer in different parts 132 .The microstructures were rinsed in an ethylenediamine solution.A highly selective Michael addition occurs only on the unreacted acrylate surface, leaving free amines on the surface of the acrylic portion of the structures but not on the methacrylate regions.Finally, the acrylic regions were selectively coated with Au and Cu, respectively (Figs. 5b−d).
In second approach, ordinary polymer structures serve as the 3D scaffolds for the printing of metallic structures.The Fourkas group reported laser writing of silver lines from a poly(vinylpyrrolidone) (PVP) thin film containing silver nanoparticles as silver cations.After printing of the polymer structure, a PVP layer was coated on its surface, followed by laser writing.In this fashion, 3D metal lines on dielectric polymer structures were realized 133 .
As discussed above, the most common approach to obtain metals by multi-photon 3D laser printing is by photoreduction of metal salts from aqueous solution.The water can also be replaced by a hydrogel, acts both as a container and 3D supporter for metal reduction (see Fig. 5e) 134 .
As an original part of this review article, we have recently realized the fabrication of isolated metal cables and truly 3D metal lines.First, 2D or 3D polymer substrates have been written by using the (electrically isolating) PETA photoresist.After development, the structure was exposed to aqueous ammonium tetrachloroplatinate 55 .Second, platinum lines were printed on the resulting surfaces (or on the glass substrate surface).A continuous 3D platinum line running over a PETA bridge is shown in Fig. 5g.As the laser impinges through the glass substrate, the refractive index of which is roughly that of the aqueous solution, metal lines can be printed underneath the polymer bridge as well as on top of it.Here we have used a water-immersion microscope objective lens (Zeiss, LD C-Apochromat 63x/1.15).The metal can also be isolated electrically to form a microcable (Fig. 5f).Here, a small gap between the metal and the polymer has developed due to polymer shrinkage.Such electrically isolated structures might be useful for applications in which the cable is used in an electrolytic environment, where contact of the metal line to the environment is unwanted.However, the shrinkage-induced gap would have to be filled-in by an additional lithography step to avoid electrical short circuits.

Multiple material properties from a single photoresist
Multiple material properties can be obtained by applying a stimulus to a given dedicated photoresist during the 3D printing process.The underlying principle is that the stimulus influences the multi-photon-absorption induced chemical reactions such that the emerging material properties can be determined locally in 3D.Here, we review the few examples in the literature which have used this approach.
The simplest possibility to control the material properties is to change the monomer cross-linking density by changing the laser exposure dose.The latter can be realized by changing the incident laser power, the focus scanning speed, by multiple exposures (connected to the hatching and slicing distance), or by combinations of these.This approach has also been coined gray-tone lithography.An example for a parameter that can be changed significantly along these lines is the thermal expansion coefficient of the polymer 31 .A single photoresist (IP-Dip) was used.By changing the local exposure dose by about a factor two, the local (positive) thermal expansion coefficient could be changed by about a factor of two.In this manner, 3D metamaterials composed of two different constituents were realized, allowing to flip the sign of the effective thermal expansion coefficient.In this complex 3D arrangement, the bending of bi-material beams was translated into a checkerboard arrangement of rotations and hence resulted in a structure shrinkage (Fig. 6a).
Along these lines, the Young's modulus, the Poisson's ratio, and viscoelasticity can also be controlled 135 .Lemma et al. quantitatively analyzed commercial photoresists (Fig. 6b) including radical-initiated photo-cross-linked resists (IP series), a hybrid ceramic/polymer photosensitive resist (Ormocomp), and an epoxy-based resist (SU-8 2100).On this basis, cylindrical polymeric structures with controlled Young's modulus and linear stiffness gradients were realized in a study of the invasion and migration of human colorectal adenocarcinoma (LS-174T) tumor cells with complex mechanical properties 136 .
A closely related approach uses pNIPAM (see above) 119 .It should be noted though that this approach only works under aqueous conditions, whereas ref31.used ambient conditions.Differently designed responses were achieved using temperature and light as stimuli (Fig. 6c, d).Such approaches may find applications in 4D printing 137 , microrobotics 138 , and microsensors 139 .
The local cross-linking density also determines the local polymer shrinkage during development and thereby the resulting local internal stress.In this manner, controllable residual stresses and programmable self-bending behavior of microstructures can be realized.Bauhofer et al. demonstrated the fabrication of bio-inspired objects with complex geometries and precisely controllable shape morphing 140 .∆n = 0.01 Optical properties can also be modulated by controlling the cross-linking density of the photoresist.Dottermusch et al. systematically investigated the influence of the exposure dose on the refractive index of the commercial photoresist IP-Dip.A refractive-index difference of up to was observed when changing the laser power from a minimum value to close to the explosion threshold 141 .The change in cross-linking density is also accompanied by a small volume change 142 .
Duan's group combined exposure-dose modulation with pH-responsive materials consisting of an acrylic acid (AAc) and N-isopropylacrylamide (NIPAM) as monomers, dipentaerythritol pentaacrylate (DPEPA) as cross-linker, 4,4'-bis (diethylamino) benzophenone (EMK) as photoinitiator, triethanolamine (TEA) as photosensitizer, and PVP as viscosity promoter 137 .The final hydrogel structures exhibited excellent pH-responsive properties and were also sensitive to other stimuli, such as chemical solvents and temperature.By again controlling the local exposure dose, shape-morphing of the final structure was achieved.Rapid, precise, and reversible 3D-to-3D shape transformations for the construction of complex 3D reconfigurable compound micromachines were demonstrated (Fig. 6e).Similarly, Ling's group employed another kind of BSA-based pH-responsive material.By controlling the cross-linking density via the slicing distance at various regions with the same structure, the swelling extent of the microstructures can be quantified and controlled with high precision and anisotropic responsiveness across a single BSA structure can be achieved.For example, by patterning microstructures with alternating low and high cross-linking section, reversible geometrical circle-to-polygon and polygon-to-circle shape morphing was achieved under pH changes 26 .A freestanding 3D microtrap which is able to open and close in response to a pH change was also demonstrated 27 .
Other examples include programming of the exposure dose by setting laser scanning path and scanning steps in solvent responsive photoresist, which enables reversible deformation and actuation of microstructures using different solvents 138 , or by fabricating ion-responsive microcantilevers using ionic responsive photoresist in a "bimaterial" based-design, leading to large bending angles of −33° within one second upon transferring the microcantilevers from water to sodium chloride solution 139 .
The wavelength of the writing laser is another promising stimulus to modulate the material properties during 3D printing using a photoresist that is composed of two different subsystems susceptible to different wavelengths of light.In the ideal referred to as wavelength orthogonality, one wavelength leads to material property #1 and a second wavelength to material property #2.Even without such ideal orthogonality, the two wavelengths may result in significantly different properties.While several material systems have been reported based on one-photon polymerization process 143−147 , this concept has so far not been demonstrated for multi-photon-based 3D laser printing.We briefly mention though that additive manufacturing by multi-photon polymerization at one wavelength and multi-photon subtractive manufacturing at another wavelength has been demonstrated 148 .
Yet another stimulus is the writing laser optical polarization.For the SZ2080 organic-inorganic hybrid material, it has been shown that varying the polarization allows for controlling the polymerization process on a scale of nanometers, leading to the variations in width and symmetry of the 3D suspended polymerized bridges and photonic crystals under tight focusing 149 .In a liquid-crystalmonomer photoresist, well controlled laser polarization facilitated the generation of tunable polymerization patterns 150 .Furthermore, external topographical features can stimulate the alignment of the liquid-crystal director 151 .

Conclusions
We have reviewed the status of multi-material multiphoton 3D laser printing on the micrometer and nanometer scale.Fig. 7 provides a histogram of achieved combinations of dissimilar material properties reviewed here.Concerning primary materials, the field still shows shortcomings concerning electrically conductive, semiconducting, metallic, and stimuli-responsive ingredients.These can be combined to multi-material 3D architectures, e.g., by using an integrated microfluidic system.However, we have pointed out that such sequential 3D printing of dissimilar photoresists is prone to some fundamental restrictions connected to the fact that 3D laser printing fundamentally requires optically transparent photoresists or materials and that it requires small spatial modulations of the refractive index to avoid large optical aberrations during the printing process.Therefore, more recent approaches seek to use just a single (composite) photoresist and dedicated stimuli during the 3D printing process that allow defining the material properties locally within each voxel.However, these approaches are in their infancies and it is presently not clear yet what the fundamental limitations are.

Fig. 2
Fig. 2 Multi-material 3D microarchitectures for biological studies and applications.a Top-view optical fluorescence image of a two-component scaffold with fibronectin-coated cell adhesion points (red), cell-repellent parts (gray), and cells grown within (green) [obtained from ref. 89].b, c Electron micrograph and optical fluorescence image, respectively, of a related 3D scaffold structure [obtained from ref. 109].d Multi-protein microstructures for cell migration and adhesion enhancement [obtained from ref. 90].e Multi-component 3D microscaffold with 3D spatially defined proteinaceous environments [obtained from ref. 111].f 3D scaffold containing surfaces functionalized by two different extracellular matrix proteins and one cell-repellant polymer [obtained from ref. 92].g -i 3D stimulusresponsive multi-material based scaffold serving as a micro-stretch-bench for cells (green), specifically adhering to the red regions in the scheme g.The yellow hostguest hydrogel in the middle of g swells reproducibly and thereby bends the elastic polymer lamellae [obtained from ref. 113].

Fig. 3
Fig. 3 Multi-material 3D microarchitectures for applications in optics and photonics.Left: Scheme of a 3D deterministic fluorescent security feature containing four differently doped polymers emitting at four different wavelengths (red, blue, green, and yellow) and one non-fluorescent polymer component.Right: Measured fluorescence stack [obtained from ref. 105].b Colored electron micrograph.A microoptical lens composed of two different polymers (blue and green) has directly been 3D printed onto the end facet of a single-mode optical fiber (red) [obtained from ref. 115].c Polymer waveguide on a substrate and ring liquid-crystal elastomer optical ring cavity.By stimuli such as temperature and light, the liquid-crystal elastomer swells/contracts, allowing for tuning the coupling between waveguide and resonator [obtained from ref. 116].

Fig. 4
Fig. 4 Multi-material 3D microarchitectures for applications as microactuators and microrobots.a Scheme of a structure composed of the protein BSA and an SU-8 skeleton.b Corresponding experimental data showing such "artificial muscle " [obtained from ref. 103].c The stimulus responsiveness of LCE combined with a passive polymer (here IP-dip) allows for obtaining bending behavior, by which a walker can move [obtained from ref. 128].d Related architecture including two differently oriented liquid-crystal elastomers plus passive IP-dip "nails ".Each stripe has a size of 20 × 200 × 20 μm 3 .This structure behaves as a reproducible gripper [obtained from ref. 127].

Fig. 5
Fig. 5 Multi-material 3D microarchitectures including conductive parts for applications in microelectronics.a Side-by-side silver-coated and bare polymer horseshoe structures made by selective electro-less plating of two different polymers [obtained from ref. 88].b−d Examples for selectively copper-coated 3D structures.Each link in b is 70 μm long.Adjacent polymer squares in c are 20 μm on a side [obtained from ref. 132].e Cross-grid structure manufactured by multiphoton induced-reduction of two different metal salts in a hydrogel.The vertical lines (red) are made out of gold, the horizontal ones (yellow) out of silver [obtained from ref. 134].f, g Colored electron micrographs of two different 3D hetero microstructures composed of an electrically isolating polymer (PETA, yellow) and conductive platinum (pink) made by multi-photon reduction.The structure shown in f can be seen as an isolated electrical cable on the micrometer scale.The structure in g demonstrates that the electric wires can be guided towards the third dimension by using an ordinary polymer as support structure.Original results by the authors of this paper.

Fig. 6
Fig. 6 Multi-material 3D microarchitectures made by using a single stimulus-responsive multi-photon photoresist.The structures in this gallery are manufactured by applying different stimuli to a photoresist during the manufacturing process, leading to dissimilar material properties in the final multi-material 3D structure.a metamaterial exhibiting a negative effective thermal expansion coefficient.The two different positive thermal expansion coefficients of the differently cross-linked polymer material are different by nearly a factor of two.They are realized by varying the laser exposure dose during the 3D printing [obtained from ref. 31].b Measured Young's modulus, , of 3D printed polymers versus laser exposure power at otherwise fixed printing parameters.Depending on the used photoresist (see legend), the Young's modulus can be changed by more than a factor of three [obtained from ref. 135].c Bimaterial beams exhibiting pronounced bending actuation made similar to the structure in a, but using a hydrogel.d Snapshots of several of these beams with calculated temperature profile overlaid onto one of them.In an aqueous environment, the effects are much larger than the ones under ambient conditions in a [obtained from ref. 119].e Examples of 4D reconfigurable compound micromachines, including a microstent, a microcage, and a microumbrella.Again, the mechanical properties of the different hydrogel parts are tailored by changing the local cross-linking density via the laser exposure dose [obtained from ref. 137].
Forschungsgemeinschaft (DFG, German Research Foundation) under Germany 's Excellence Strategy for the Excellence Cluster "3D Matter Made to Order" (EXC 2082/1-390761711), by the Carl Zeiss Foundation, by the Helmholtz program "Materials Systems Engineering (MSE) ", and by the Karlsruhe School of Optics and Photonics (KSOP).

Fig. 7
Fig.7Histogram of references reviewed in this paper addressing structures made by multi-photon 3D laser printing containing dissimilar ingredient material properties.For example, 9 papers reviewed here have presented multi-material architectures containing at least two different dielectric materials (entry dielectric-dielectric in this matrix).References may appear twice.For example, a stimulus-responsive material can at the same time be magnetic or elastic.The material properties are ordered according to the number of references occurring along the diagonal of this matrix.As the matrix is symmetric by definition, the redundant part is left away.The numbers in brackets on the side of the bars refer to the references summarized by the respective bar.Clearly, while some materialproperty combinations have been realized in several publications, several other material-property combinations have not so far.