KEYWORDS: Printing, Two photon polymerization, Standards development, 3D microstructuring, Microfluidics, Microfabrication, In vivo imaging, Biomedical applications, Artificial intelligence, Tissue engineering
Herein, we demonstrate the translation of Two-Photon Grayscale Lithography (2GL®), as well as Aligned 2-Photon Lithography (A2PL®), to biomedical applications. Specifically, we will present a novel workflow of aligned two-photon polymerization (2PP) microfabrication for 3D cell assays and perfusion inside microfluidic devices. For completeness, we also reveal how 2GL® can be applied to artificial intelligence (AI) generated topographies for enhanced and scalable 2.5D cell culturing. The versatility offered by both aligned and 2GL® printing holds great promise for various applications in biotechnology, tissue engineering, and microfluidics, creating new opportunities for innovation within established biomedical and pharmaceutical industries.
Measuring molecular orientation properties is very appealing for scientists in molecular and cell biology, as well as biomedical research. Orientational organization at the molecular scale is indeed an important brick to cells and tissues morphology, mechanics, functions and pathologies. Recent work has shown that polarized fluorescence imaging, based on excitation polarization tuning in the sample plane, is able to probe molecular orientational order in biological samples; however this applies only to information in 2D, projected in the sample plane. To surpass this limitation, we extended this approach to excitation polarization tuning in 3D. The principle is based on the decomposition of any arbitrary 3D linear excitation in a polarization along the longitudinal z-axis, and a polarization in the transverse xy–sample plane. We designed an interferometer with one arm generating radial polarization light (thus producing longitudinal polarization under high numerical aperture focusing), the other arm controlling a linear polarization in the transverse plane. The amplitude ratio between the two arms can vary so as to get any linear polarized excitation in 3D at the focus of a high NA objective. This technique has been characterized by polarimetry imaging at the back focal plane of the focusing objective, and modeled theoretically. 3D polarized fluorescence microscopy is demonstrated on actin stress fibers in non-flat cells suspended on synthetic polymer structures forming supporting pillars, for which heterogeneous actin orientational order could be identified. This technique shows a great potential in structural investigations in 3D biological systems, such as cell spheroids and tissues.
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