Sofía Rodríguez, product marketing manager, Nanoscribe
Two-photon polymerisation (2PP) is a high-precision 3D printing technology employed for numerous microfabrication applications in various fields, ranging from biomedical engineering, microoptics and micromechanics to metamaterials and microfluidics. The enormous potential of this technology lies in its inherent design freedom, sub-micrometre resolution and extensive range of printing materials. It is therefore finding new use scenarios in research, prototyping and production processes. In particular, the variety of printing materials has expanded considerably in recent years, enabling the development of novel devices possessing specific chemical, biological, mechanical and optical properties. Together, the 2PP printer hardware, software and printing materials offer a complete and efficient solution for many challenging 3D microfabrication tasks.
2PP emerged over two decades ago and pushed 3D microfabrication forward in terms of design freedom and versatility. In 1997, Shoji Maruo and Satoshi Kawata experimentally demonstrated the principle of 2PP1. Ten years later, Nanoscribe, a spin-off of the Karlsruhe Institute of Technology (KIT), commercialised this technology that today is used for a multitude of high-precision 3D microfabrication applications on the nano-, micro- and meso-scale. Since the foundation of Nanoscribe, the number of 2PP adopters in research and industry has increased year by year.
Scientists and engineers in R&D organisations and industry are using 2PP for pioneering work, such as the fabrication of microscopic implants, scaffolds for tissue engineering, microfluidic mixers, microneedles and beam steering microoptics. The printing materials play a significant role in the feasibility of best quality designs. They define the intended final qualities of the printed object, such as resolution, surface roughness, specific chemical, mechanical or optical properties, and printing time. To this effect, R&D activities are as important for printing materials as they are for high-end systems to achieve even more powerful 2PP capabilities.
The technology
Two-photon polymerisation (2PP)—also referred to as multiphoton polymerisation, multiphoton lithography or direct laser writing—belongs to a family of additive manufacturing (AM) technologies that use light to cure a liquid photoresin and thus create a digitally defined 3D microstructure. Typically, UV light is used to cure the photoresin2. However, 2PP uses lower energy light, namely in the near-infrared (NIR) range. This causes the photoresin to solidify only if its molecules simultaneously absorb the energy of two photons, a process known as two-photon absorption. This event is only likely to happen in the focal volume of pulsed light because it requires a high intensity inside a volume of the photosensitive material. Light and a molecular component, the photoinitiator, trigger a chemical reaction in the photoresin. Upon excitation, the monomers in the liquid photoresin convert to a cross-linked solid state. As a result of this reaction, a solid, insoluble thermoset polymer forms within the exposed resin volume.
A galvanometer scanner moves the focal point in the focal plane at speeds of hundreds of millimetres per second, enabling fast and precise printing. In addition, a stage system that has nanopositioning capabilities in all three dimensions moves the substrate and the photoresin to build up the 3D microstructures layer by layer. Software plays a key role in ensuring efficient printing by controlling all printer hardware and parameters throughout the process. This enables a straightforward workflow from importing a CAD model to printing the microstructures.
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The galvanometer scanning mirrors steer the light and cure the photoresin only in a tight laser focus, thereby creating a 3D microstructure.
The essential role of printing materials
The right choice of printing material is decisive in 2PP, as it is in all 3D printing technologies. The properties of a printing material determine quality, function and performance of the final printed microstructures.
Thermosetting polymers (thermosets) are the dominant material class for 2PP. The polymer chains in a thermoset are strongly cross-linked and thus form an infusible and insoluble network. The most common chemical mechanism for thermoset curing in 2PP is light-stimulated radical polymerisation.
2PP photoresins must be optimised for and tailored to specific applications. This means that they are ready for immediate use and designed to achieve the desired results. In addition, 2PP photoresins should have a large dynamic range so that they can offer the flexibility necessary to further improve the features and qualities of printed microstructures and objects. The dynamic range starts with the minimum exposure dose required for curing and extends to the maximum exposure dose before the structure deteriorates. A large dynamic range allows for a wide selection of printing parameters and paves the way for ideal printing results to fulfil different requirements.
As radical polymerisation is a well-understood process, scientists and engineers investigate many 2PP-compatible custom materials and process variations. The printed objects can thus gain additional functions such as responsive qualities3 and enable the development of innovative bioapplications4.
Biocompatible resins for cell studies
One of the application fields that 2PP is being used in is tissue engineering. The study of cells and their interaction with neighbouring cells, the extracellular matrix and surrounding molecules enables investigations in areas ranging from stem cell research and drug screening to regenerative medicine. However, the natural complexity of these interactions requires the mimicking of the cells’ surrounding microscopic environments in all three dimensions.
Scientists and engineers are searching for new 3D microfabrication methods and materials that meet the requirements of cell culture platforms. Advances in 2PP have demonstrated its potential for the fabrication of complex microstructures such as cell scaffolds and membranes. Moreover, the variety of 2PP photoresins for cell scaffolds is increasing and revealing insights into how different synthetic microenvironments can influence cell growth, proliferation and differentiation.
The biocompatibility of materials used in 2PP is decisive for the desired performance of 3D printed cell scaffolds. Nanoscribe has a broad portfolio of (meth)acrylate-based 2PP photoresins and some of these provide the biocompatibility required for cell cultures. They have undergone ISO 10993–5 Biological evaluation of medical devices—Part 5: tests for in vitro cytotoxicity, proving their suitability for 3D printed cell-friendly scaffolds, and can realistically mimic microenvironments.
Scientists at the University of Iowa are studying 3D printed retinal cell scaffolds for tissue engineering. The retinal cells have been derived from induced pluripotent stem cells (iPSCs) and seeded onto cell scaffolds made of biocompatible 2PP photoresins5. iPSCs and iPSC-derived cells are known to be very sensitive to their environment and very difficult to culture; however, after two days of culturing, the retinal cells and vertical pores of the scaffolds had aligned.
One of Nanoscribe’s biocompatible photoresins is called IP-Visio. It exhibits very low autofluorescence, thus providing a clear view through the printed scaffolds in fluorescence microscopy images. This enables scientists to analyse cellular components and processes unhindered by the printed microstructures.
Biopolymer cell scaffolds
2PP is also compatible with a broad range of polymers, including those that are naturally occurring. These can mimic the biochemical and biomechanical properties of natural tissues6, 7.
Scientists at the University of Iowa have also been investigating the use of four biopolymer 2PP photoresins for biopolymer cell scaffolds, namely methacrylate-modified collagen, gelatin, hyaluronic acid (HA) and polycaprolactone (PCL). The biocompatibility of these photoresins has been confirmed according to ISO 10993 and through the successful cultivation of different cell types on 3D printed cell scaffolds over several days. In addition, the PCL scaffolds, which carry photoreceptor cells, have been transplanted into the sub-retinal space of pigs for more than 30 days. There has been no evidence of retinal injury, inflammation, systemic toxicity or tumour formation during this period.
The capabilities and versatility of 2PP in the fabrication of such porous scaffolds make it a suitable technology for use in critical applications, for example, the restoration of neural function in patients suffering from neurodegenerative diseases.
A cell scaffold printed with the IP-Visio photoresin.
Nanostructured microoptics
2PP facilitates the realisation of extremely compact and accurate 2D, 2.5D and 3D microoptics in complex shapes. The latest advances in 2PP allow for the integration of diffractive features into refractive microlenses to create hybrid microoptical elements. These are miniaturised components that enable the manipulation and detection of light in miniaturised sensing devices and integrated photonics. Microoptical elements are integrated into compact devices such as mobile phones to provide additional functionality.
2PP can also be used for the in-situ fabrication of high-precision optics on various substrates, including pre-patterned photonic integrated circuits, single- and multi-mode fibres, laser chips and wafers. This direct printing approach can help to integrate microoptics in multiple applications, including automotive lighting, consumer electronics, endoscopy devices and metrology equipment.
There is a host of 2PP photoresins available for microoptics. They enable different qualities to be achieved depending on the application, for example, the combination of a high-resolution photoresin and use of a high-numerical aperture objective lens allows for the realisation of extremely fine nanostructured diffractive optical elements (DOEs). This means that multi-level DOEs can provide the high resolution, submicron lateral and axial features required to structure light by diffraction as well as generate patterns of dots, grids or even complex images in the far field. DOEs are mostly used in applications that require structured light such as 3D facial recognition on smart devices.
Another advantage of 2PP is that it allows for the fabrication of multi-level DOEs in one printing step, unlike other time-consuming and costly processes involving multiple lithographic, etching and alignment steps. Moreover, up to 4,096 level designs can be processed efficiently and cost effectively via discrete or quasi-continuous topographies.
A compound lens system with two refractive elements printed with the IP-n162 photoresin. The printing material has a high refractive index of 1.62, high dispersion and low infrared absorption. Printed by Nanoscribe, optical design by Simon Thiele, TTI, TGU Printoptics.
High optical quality refractive microlenses
2PP is also an efficient and cost-effective fabrication method for the refractive microoptics used in beam shaping, collimation, light homogenisation, illumination and imaging applications. Spherical, aspherical and even freeform microoptical designs can be produced. The 2PP process enables the fabrication of microlenses in single elements, regular arrays and random arrangements that are useful in, for example, homogeniser components for illumination applications.
A 2PP photoresin called IP-S is well suited for the printing of microstructures with high shape accuracy, very smooth contours and optical quality surfaces. For example, microlens arrays (MLAs) printed with the IP-S achieve a shape accuracy better than 1 µm and a surface roughness of less than 10 nm Ra.
A recently introduced 2PP photoresin called IP-n162 has a high refractive index of 1.62 and high Abbe number (dispersion measurement) of 25. The optical properties of IP-n162 are comparable to those of optical polymers typically used in injection moulding, for example, polycarbonates and polyesters. It can therefore be used for prototyping of microoptics, enabling the avoidance of time-consuming and expensive diamond-milled injection mould iterations.
IP-n162 is suitable for high-performance refractive or hybrid microoptics in a wide range of applications, for example, miniaturised imaging and 3D sensor systems for augmented reality (AR) and virtual reality (VR) applications. It also paves the way for compound microoptics consisting of elements that have different dispersions, for example, IP-n162 and the lower refractive index resin IP-S can be a perfect match for creating achromatic microoptical systems.
Another advantage of IP-n162 is that it has low absorption in the infrared spectrum. It has a 10 times lower absorption than IP-S in the 1,200 to 1,550 nm wavelength range. This makes IP-n162 a suitable material for applications in infrared optics, optical communication and photonics packaging, where low absorption losses are critical.
Glass microoptical elements
Fused silica glass is being explored as a 3D printing material. It has excellent optical, chemical and mechanical properties that remain stable in the long term. However, glass is extremely difficult to manufacture. Nanoscribe and Glassomer are collaborating under the European research project OptoGlass3D on the development of a printing process for 3D microoptical elements in fused silica glass. The printing process is based on 2PP and incorporates Glassomer’s glass shaping technology. The companies seek to validate the process by showcasing the fabrication of fused silica glass microcomponents. They also intend to demonstrate the ability to tune microoptical properties as required for future applications in imaging, sensing and photonics.
Nanoscribe offers a broad portfolio of photoresins specifically designed for 2PP. These materials provide different printing and physical properties, for example, for slicing the object volume in fine layers (slicing distance) and filling each layer with lines (hatching distance).
Conclusion
The compatibility of 2PP with many advanced printing materials increases its versatility, opening up new applications. It allows for the rapid creation of microstructures of practically any shape and with various properties. Scientists and engineers are able to design and iteratively create microstructures with varying parameters or completely different shapes without additional costs. In addition, 2PP offers the design freedom to fabricate freeform, porous and even organic geometries with high shape accuracy. A digital workflow from CAD design to printed part means that the technology can be applied to various plane and pre-defined substrates, including photonic chips, optical fibres and wafers.
Finally, there is the question of how 2PP can be scaled up for mass production. To solve this issue, the 3D- printed microstructures can also serve as masters for subsequent replication processes in micromanufacturing. 2.5D polymer masters have been demonstrated to fit into standard mass replication processes such as injection moulding, (direct) hot embossing and nanoimprint lithography.
Nanoscribe
References
1Maruo, S., Nakamura, O. and Kawata S. (1997). Three-dimensional microfabrication with two-photon-absorbed photopolymerization. Optics Letters, volume 22, issue 2, pp.132–134.
2Nanoscribe (2020). Additive-manufacturing based on two-photon polymerization—a key enabling technology for high-precision 3D printing [whitepaper]. Available at: https://bit.ly/35TPmCY
3Hippler, M., Blasco, E., Qu, J., Tanaka, M., Barner-Kowollik, C., Wegener, M. and Bastmeyer, M. (2019). Controlling the shape of 3D microstructures by temperature and light. Nature Communications, volume 10, article number 232, pp.1–8.
4Liao, C., Wuethrich, A. and Trau, M. (2020). A material odyssey for 3D nano/microstructures: two-photon polymerization-based nanolithography in bioapplications. Applied Materials Today, volume 19, article number 100635.
5Worthington, K. S., Wiley, L. A., Kaalberg, E. E., Collins, M. M., Mullins, R. F., Stone, E. M. and Tucker, B. A. (2017). Two-photon polymerization for production of human iPSC-derived retinal cell grafts. Acta Biomaterialia, volume 55, pp.385–395.
6Shrestha, A., Allen, B. N., Wiley, L. A., Tucker, B. A. and Worthington, K. S. (2020). Development of high-resolution three-dimensional-printed extracellular matrix scaffolds and their compatibility with pluripotent stem cells and early retinal cells. Journal of Ocular Pharmacology and Therapeutics, volume 36, issue 1, pp. 42–55.
7Thompson, J. R., Worthington, K. S., Green, B. J., Mullin, N. K., Jiao, C., Kaalberg, E. E., Wiley, L. A., Han, I. C., Russell, S. R., Sohn, E. H., Guymon, C. A., Mullins, R. F., Stone, E. M. and Tucker, B. A. (2019). Two-photon polymerized poly(caprolactone) retinal cell delivery scaffolds and their systemic and retinal biocompatibility. Acta Biomaterialia, volume 94, pp.204–218.