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

Abstract

Non-linear excitation microscopy offers a number of advantages for in vivo imaging compared to conventional confocal techniques. However, tissue penetration can still be an issue due to both scattering and spherical aberrations induced on highly 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 the optical aberrations. Here, we develop and test, on a monolayer of fibroblast cells, plano-convex micro-lenses 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 wave front of the lenses is reported together with their modulation transfer function and wave front profile. Notably, we could retrieve magnified fluorescence images through the microlenses coupled to commercial confocal and two-photon excitation raster 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 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.

Two-photon laser polymerized microlenses for nonlinear excitation microscopy of biological samples

“…This procedure leads to a 98% reduction in the fabrication time with respect to volume polymerization, still obtaining sufficient optical quality to use the lenses for direct wide-field imaging of cells, both in transmission and in fluorescence mode. We also show that the microlenses can be simply coupled to a raster scanning non-linear excitation optical microscope, even by using low numerical aperture and low magnification objectives in a virtual image coupling configuration [5]. By changing the distance between the microscope objective and the microlenses we can tune the depth of focal plane in the sample to which the microlenses are directly coupled.…”

High-Transparency 3D Micro-Optics of Hybrid-Polymer SZ2080™ Made via Ultrafast Laser Nanolithography and Atomic Layer Deposition

“…Micro-spectrometers [32] and holograms [33], as well as non-linear imaging applications [34] where compact dimensions are well integrated with multiple functionalities may also obtain considerable improvement.…”