Microlenses Fabricated by Two‐Photon Laser Polymerization for Cell Imaging with Non‐Linear Excitation Microscopy

Abstract

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, Fibroblast cell culture plano‐convex microlenses to be used for non‐linear imaging of biological tissue are developed and tested. 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. Magnified fluorescence images can be retrieved 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.

Two-photon polymerization lithography for imaging optics

“…The unique ability to achieve complex optical imaging systems with self-alignment in one-step manufacturing while maintaining nanoscale precision makes it outperform other technologies with special convenience for global optimization design and fast integration. Coupled with the rapid advancements in materials and fabrication techniques, TPL is ready to replace traditional fabrication methods in specific imaging applications and explore new directions and fields for imaging with compact configurations and outstanding optical performance [169,183,[264][265][266][267][268]. We envision that the future development will promise to reduce the cost significantly, facilitating the transition of this technology from the laboratory to industry for the mass production of more reliable and complex imaging systems and applications.…”

“…Aside from CEs, Schäffner et al demonstrated a spherical MLA to generate multiple optical tweezers patterns that are individually controlled by spatial light modulation from a digital micromirror device [167,168]. Moreover, Marini et al demonstrated MLAs for non-linear excitation microscopy of biological samples and in-vivo inspection of cellular dynamics (figure 13(e)) [169]. Finally, MLAs fabricated by TPL have been used for spectroscopic measurements, where Bogucki et al demonstrated aspherical MLAs on single light emitters such as quantum dots and van der Waals heterostructures [170].…”

Review of optical methods for noninvasive imaging of skin fibroblasts—From in vitro to ex vivo and in vivo visualization

“…[83]. fibroblasts in vivo [110]. Human skin fibroblasts recorded in vitro by TPE-FM are shown in Figure 4.…”

Reconfigurable Microlens Array Enables Tunable Imaging Based on Shape Memory Polymers

“…In recent years, the advances of micro-/nanofabrication technologies , have pushed forward the rapid progress of the manufacturing methods for miniature optical devices. Various types of MLAs can be successfully fabricated through advanced additive and subtractive manufacturing techniques. − For example, photolithography hot melting, self-assembly, − 3D printing, , photon aggregation, , and inkjet printing , have been successfully employed for producing MLAs with different geometrical configurations and chemical compositions. For example, diamond turning enables preparing microlenses with desired surface profiles on hard materials such as monocrystalline silicon and glass. , The combined technology that involves laser processing and dry/wet etching has also been developed for MLAs. , As a pioneer of this field, Chen et al have prepared various MLAs and CEs by combining laser processing-assisted wet etching with soft lithography and hot embossing. , To promote the fabrication efficiency and guarantee the uniformity of CEs, Liu et al directly prepared templates for CEs on a curved sapphire substrate using laser processing combined with dry etching .…”