3D printing, also known as ”additive manufacturing”, indicates a set of fabrication techniques that build objects by adding material, typically layer by layer. The main advantages of 3D printing are unlimited shapes and geometry, fast prototyping, and cost-effective small scale production. Two-photon lithography (TPL) is a laserbased 3D printing technique with submicron resolution, that can be used to create miniaturized structures. One of the most compelling applications of TPL is the 3D printing of miniaturized optical elements with unprecedented complexity, small-scale and precision. This could be potentially beneficial in biophotonics, a multidisciplinary research field in which light-based techniques are used to study biological processes. My research has been aimed at demonstrating novel applications of 3D printing based on TPL to different biophotonic applications. In particular, here we show 3D printed micro-optical structures that enhance and/or enable novel functions in advanced biophotonics methods as two-photon microendoscopy, optical trapping and Stimulated Raman Scattering microscopy. Remarkably, the micro-optical structures presented in this thesis enable the implementation of advanced techniques in existing or simpler microscopy setups with little to no modification to the original setup. This possibility is essentially allowed by the unique miniaturization and in-situ 3D printing capabilities offered by TPL.

Additive manufacturing using 2 photon polymerization is of great interest as it can create nanostructures with feature sizes much below the diffraction limit. It can be called as true 3D printing as it can fabricate in 3 dimensions by moving the laser spot in any 3D pattern inside the resist. This unique property is attributed to the non-linearity of two photon absorption which makes the polymerization happen only at the focal spot of the laser beam. This method has a wide range of applications such as optics/photonics, metamaterials, metasurfaces, micromachines, microfluidics, tissue engineering and drug delivery.<br>This work focuses on utilizing 2 photon fabrication for creating a metasurface by printing diabolo antenna arrays on a glass substrate and subsequently metallizing it by coating with gold. A femtosecond laser is used along with a galvo-mirror to scan the geometry inside the photoresist to create the antenna. The structure is simulated using ANSYS HFSS to study its properties and optimize the parameters. The calculations show a reflectance dip and zero reflectance for the resonance condition of 4.04 μm. An array of antennas is fabricated using the optimized properties and coated with gold using e-beam evaporation. This array is studied using a fourier transform infrared spectrometer and polarization dependent reflectance dip to 40% is observed at 6.6 μm. The difference might be due to the small errors in fabrication. This method of 3D printing of antenna arrays and metallization by a single step of e-beam evaporation is hence proved as a viable method for creating optical metasurfaces. Areas of future research for perfecting this method include incorporating an autofocusing system, printing more complicated geometries for antennas, and achieving higher resolution using techniques like stimulated emission depletion.

We characterize the impact of high-energy, 172 nm vacuum ultraviolet photons on the molecular weight and the glass transition temperature of poly(methyl methacrylate). We found that the molecular weight is reduced strongly on the surface of the exposed samples with a continuous transition towards the unexposed bulk material being located below the modified region. The glass transition temperature was found to be significantly lowered in the exposed region to well below 50°C compared to that of the 122°C of the bulk region. We could use this material contrast to selectively reflow the top surface of the exposed samples only. This allowed us to create ultra-smooth micro-optical structures by postprocessing without influencing the overall geometry that is required for the optical functionality.