Fabrication of Plano-convex Microlenses using Two-Photon Polymerization for Bioimaging with Non-Linear Excitation Microscopy

. A recent challenge in bioimaging is the observation and imaging of vital, thick, and complex tissues in real time and in non-invasive mode. In the last decade, non-linear excitation microscopy showed several advantages for in-vivo imaging compared to conventional confocal techniques. Nevertheless, deep tissue imaging remains challenging, especially for thick media, due to spherical aberrations induced on focused beams by the tissue. A low numerical aperture objective lens coupled to high dioptric power microlenses, implanted in the tissue, can be beneficial for the reduction of optical aberrations. In this context, we fabricated a system of plano-convex microlenses and microscaffolds on a single chip by means of two-photon polymerization), to be used for non-linear imaging of biological specimens.


Introduction
One of the fascinating advancements in in-vivo imaging is the possibility to observe live tissue.Today, nonlinearexcitation microscopy in NIR (800-1600 nm) can increase the penetration depth by reducing the effects of scattering on the point spread function [1,2].However, the spatial resolution is also low in deep tissue imaging due to spherical aberrations.The use of low numerical aperture objectives to pass through the outer layers of the tissue, together with high dioptric power microlenses implanted in-vivo close to the observation volume, can allow in-vivo imaging with reduced spherical aberration [3].
Here we report on the design, fabrication, and validation of plano-convex microlenses for two-photon excitation microscopy.These lenses, fabricated by two-photon polymerization (2PP) in a biocompatible photoresist (SZ2080), can be used individually or multiplexed in an array [3,4].The dioptric power and magnification of microlenses were quantified by coupling them directly with a low numerical aperture objective in a raster scanning microscope, to image stained human fibroblasts.In addition, a system of microlenses, combined with microscaffolds [4], was developed as a single chip for non-linear imaging.

Results and Discussion
The plano-convex microlens is designed and optimized with Zemax, resulting in a lens with diameter 282 µm, curvature radius 147 µm, and thickness 160 µm.Then, the microlens is fabricated on top of the supporting borosilicate glass with thickness of 170 µm.We developed a protocol which allows to fabricate a planoconvex lens in 8 minutes using the combination of 2PP and UV exposure (Fig. 1-A).The fabrication process was carefully optimized to obtain the smoothest surface for the microlens (Fig. 1-B).The focal length of the fabricated lens was measured with optical characterization (Fig. 1-C).We could retrieve magnified fluorescence images of stained cells cultured in vitro through the microlenses coupled to a commercial two-photon excitation scanning microscope (Fig. 1-D).The signal-to-noise ratio of the images acquired through the fabricated microlens is not substantially affected by the use the microlenses [3].The magnification and focal plane can be adjusted by changing the relative position of the microlenses with respect to the microscope objective and the immersion medium.We also designed and developed a dedicated protocol to fabricate a chip (Fig. 2-A) composed of a system of microlens (D = 282 µm, and R= 147 µm ) (Fig. 2-B), and a 3D microscaffold (400 × 400 × 100 ), (Fig. 2-C) which is also fabricated by 2PP in SZ2080 on top of the same borosilicate glass with thickness of 170 µm.

Conclusion and Outlook
The results demonstrate that the use of a microlens as a single lens, or as a microfabricated chip (a lens coupled to a scaffold) allows the reduction of spherical aberrations in in-vivo imaging.This work opens the way to implanting micro-optical devices for the direct and continuous optical inspection of biological dynamics in vivo.

Fig. 1 .
Fig. 1. (A) SEM image of one plano-convex lens.(B) Close-up of the central region on the top plane of the lens.(C) Optical characterization of one lens (f = 44 µm).(D) Fluorescence images of cells under two-photon excitation through a single microlens.Fabrication was performed by a femtosecond laser with wavelength  =1030 nm, 280 fs pulse duration, and 1 MHz repetition rate, and the beam was tightly focused by an objective 100x, W with NA= 1.1 onto the photosensitive resist.