Microlenses fabricated by two-photon laser polymerization for intravital cell imaging with non-linear excitation microscopy

Non-linear excitation microscopy offers several advantages for in-vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to scattering and spherical aberrations induced on focused beams by the tissue. The use of low numerical aperture objectives to pass through the outer layers of the skin, together with high dioptric power microlenses implanted in-vivo close to the observation volume, can be beneficial to the reduction of optical aberrations. Here, we develop and test on fibroblast cell culture plano-convex microlenses to be used for non-linear imaging of biological tissue. The microlenses can be used as single lenses or multiplexed in an array. A thorough test of the lenses wavefront is reported together with the modulation transfer function and wavefront profile. We could retrieve magnified fluorescence images through the microlenses coupled to commercial confocal and two-photon excitation scanning microscopes. The signal-to-noise ratio of the images is not substantially affected by the use of the microlenses and the magnification can be adjusted by changing the relative position of the microlens array to the microscope objective and the immersion medium. These results are opening the way to the application of implanted micro-optics for optical in-vivo inspection of biological processes.