Herein, we demonstrate the translation of Two-Photon Grayscale Lithography (2GL®), as well as Aligned 2-Photon Lithography (A2PL®), to biomedical applications. Specifically, we will present a novel workflow of aligned two-photon polymerization (2PP) microfabrication for 3D cell assays and perfusion inside microfluidic devices. For completeness, we also reveal how 2GL® can be applied to artificial intelligence (AI) generated topographies for enhanced and scalable 2.5D cell culturing. The versatility offered by both aligned and 2GL® printing holds great promise for various applications in biotechnology, tissue engineering, and microfluidics, creating new opportunities for innovation within established biomedical and pharmaceutical industries.

The 3D fabrication of micro-optics and photonic architectures using organic materials are limited due to poor thermal and chemical stability. We are working on high-resolution 3D printing of photonic components using commercial ORMOCOMP® hybrid polymers. This solvent-free, thermally stable resin allows to limit the shrinkage between 4 and 6%. In this work, we optimize the femtosecond Direct Laser Writing process and we use 4D-printing by modulating the laser intensity during the process. In this presentation we will investigate the relationship between linear optical properties and 4D printing parameters in order to print a core-clad waveguide.

We show that quantitative phase imaging (QPI) is a facile method for in-situ shape monitoring during multi-photon laser printing. In-situ QPI requires minimum hardware modifications of a commercial 3D laser printer and takes only a few seconds for data acquisition. We validate the approach by comparing in-situ phase reconstructions with independent ex-situ height measurements. Example applications are polymer refractive-index retrieval, shrinkage measurements, and shape reconstructions of printed microoptical devices such as microlenses and diffractive optical elements. Our research paves the way for routine monitoring of 2.5D printed structures and on-the-fly shape optimization of printed microoptics.

Our optical positioning and linking (OPAL) platform enables the assembly of complex 3D microstructures using optical tweezers and a biochemical linking mechanism. Advantages to OPAL include incorporating building blocks of different sizes and materials and augmentation of existing devices. Updates to system components and processes have improved the speed and accuracy of assembly. The collection of the backscattered signal using a quadrant photodiode permits classification of the number of trapped particles, which is used for automated assembly. The fully automated assembly will increase the efficiency of fabrication and is beneficial for rapid prototyping and producing larger, more complex structures.

The study interactions between tumor cells and lymph nodes (LNs), will be investigated with the development of complex 3D cell structures in a microfluidic chip by Laser Induced Forward Transfer (LIFT) technique. The lymphatic system and LNs are an integral part of our adaptive immune system and many tumors exploit lymphatic vessels to spread and colonize downstream LNs. The complex cell structures will be realized by the printing of tumor organoids and the multilayer printing of 2D layers of LN cells, initially on ECM and other substrates, and ultimately inside a microfluidic chip.

We present a multi-focus digital holography-based two-photon lithography (TPL) platform for high-resolution, large-scale 3D nanofabrication. Specifically, up to 2000 individually programmable laser foci are generated and controlled via digital holography and powered by a 1 kHz regenerative femtosecond laser amplifier, which effectively increases the fabrication rate to 2,000,000 voxels/sec with a lateral resolution of 90 nm. We have designed and fabricated a variety of complex 3D structures, e.g., mechanical metastructures, and experimentally verified their properties and mechanical performance. These results show our new platform is a powerful tool for applications in photonics, nanotechnology, and biotechnology.

The widescale implementation of two-photon polymerization (2PP) is limited primarily due to the cost of high-power femtosecond laser despite the high throughput and superior resolution capability. To reduce the threshold power requirement, a method is developed combining the effect of single photon absorption from a low-cost 532 nm nanosecond fiber laser with the two-photon absorption from an 800 nm femtosecond laser. The effects of spatial and temporal overlap, relative beam sizes, and frequency synchronization are also investigated.

Two-photon polymerization (2PP) is a well-established method for fabricating 3D photonic microstructures, allowing easy functionalization through photoresist doping. Recently, graphene oxide-doped cylindrical microresonators fabricated via 2PP exhibited a mode-cleaning effect on Whispering Gallery Mode (WGM) resonances. Numerical analysis unveiled that this effect results from non-uniform dopant distribution, leading to distinct levels of attenuation in radial order modes. In this study, we propose an approach to estimate dopant spacial distribution into the structure by analyzing the dopant effects in each individual radial order mode. The absorption coefficient in the structure is modeled as a parametrized function (e.g. Gaussian distribution), and finite-element simulations are carried out to optimize the parameters that best match the experimental data. This methodology provides crucial insights into non-uniform dopant concentration, contributing to a deeper understanding of the mode-cleaning phenomenon and guiding device performance enhancements.

We have demonstrated the co-doping of silver ions and Bi3+ in glass allowing creation of 3D structures with tuning luminescence properties from the visible to the near IR with fs DWL. The tunable luminescence arises from the fs laser triggering both the photochemistry of silver ions and the photo-redox reaction of Bi3+ forming low valence Bismuth ions (Bi2+ red emitter and Bi+ near IR emitter). Confocal hyper-spectral lifetime-resolved imaging revealed the shortening of the fluorescence lifetime of silver clusters in the presence of Bismuth revealing the energy transfer mechanism from silver clusters to Bi+.

Additive Manufacturing (AM) offers an alternative to conventional technologies for producing complex parts. Powder Bed Fusion of Metals using a Laser Beam (PBF-LB/M) is widely used for its high mechanical properties. However, thermally-driven stress-induced cracking is a challenge due to the uneven heat input. This study proposes a simulation-based approach to predict vulnerable regions in PBF-LB/M parts. Failure models for Inconel 718 were integrated into a PBF-LB/M process simulation, enabling the identification of critical locations prone to stress-induced fracture. Experimental validations confirmed the accuracy of a classical and an AM-adapted calibration method in predicting crack-prone zones. This approach enhances first-time-right manufacturing by enabling preemptive modifications.

Photodiodes are commonly used in Laser Powder Bed Fusion (LPBF) for process monitoring. However, they spatially average out features within the melt pool. We introduce Spatial Frequency Modulation Imaging (SPIFI), a technique enabling 1D spatially resolved imaging with photodiodes. SPIFI can produce high-resolution images using blackbody radiation or coherent illumination. In LPBF monitoring, we use three photodiodes at different wavelengths (780, 920, and 1070 nm) to obtain 1D SPIFI images of the melt pool temperature and reflected fusing beam. Our experimental results aim to benchmark LPBF physics models for accurate melt process simulations and serve as an in-situ process diagnostic.

Laser Powder Bed Fusion (LPBF) is a critical additive manufacturing process known for its accuracy and complexity in producing intricate parts. However, challenges like limited production speed, hot cracking, and material restrictions hinder its efficiency. This study explores the use of Multi-Plane Light Conversion (MPLC) as a beam shaping solution to improve LPBF. By applying MPLC, we achieve faster printing while maintaining high-quality parts. Comparative analysis demonstrates the superiority of MPLC-based beam shaping in enhancing process yield and manufacturing efficiency.

Projection multi-photon lithography, like all additive manufacturing techniques, requires optimization of process parameters to achieve geometrically accurate results. Determining these optimal parameters is often time-consuming. Machine learning can be used to avoid the need for experimentation by predicting optimal process parameters. A data collection scheme is presented where image analysis on optical microscope images is used to measure the dimensions of individual 2D layers printed with the projection multi-photon printing process for a range of process parameters. The dimensional accuracy of these 2D shapes is then used to train a Gaussian process regression model for forward prediction.

We present a new method for generating protein-based tissue engineering 3D scaffolds with adaptable mechanical properties. Multiphoton lithography allowed fabrication of 2/3D structures with feature sizes below 200 nm lateral and below 600 nm axial with Young’s moduli down to 40 kPa. The viability and mechanotransduction of mesenchymal stem cells within the scaffolds were shown by imaging of the cellular actin cytoskeleton and the vinculin protein density via 3D direct stochastic optical reconstruction microscopy. Surface functionalization of acrylate scaffolds was shown at the single-molecule level using Laser Assisted Protein Adsorption by Photobleaching (LAPAP). This pioneering research creates promising opportunities for customized tissue engineering applications.

Additive manufacturing enables producing shapes that would otherwise be expensive, or impossible to achieve. Stereolithography, specifically, offers highest resolution as it is primarily limited by the optical exposure system. With Hot Lithography, it is now possible to produce precise parts with industrially relevant material properties. We show resolution capabilities of laser scanning over an area of 200 mm x 100 mm, utilizing polymerization triggered by single photon absorption, and its applications in fields traditionally relying on microfabrication technologies. We elaborate, how Hot Lithography provides technological and economic opportunities in areas such as the electronics industry, fluidics, and medical devices.