Tailored sample mounting for light-sheet fluorescence microscopy of clarified specimens by polydimethylsiloxane casting

The combination of biological tissue clearing methods with light-sheet fluorescence microscopy (LSFM) allows acquiring images of specific biological structures of interest at whole organ scale and microscopic resolution. Differently to classical epifluorescence techniques, where the sample is cut into slices, LSFM preserves the whole organ architecture, which is of particular relevance for investigations of long-range neuronal circuits. This imaging modality comes with the need of new protocols for sample mounting. Gel matrix, hooks, tips, glues, and quartz cuvettes have been used to keep whole rodent organs in place during image acquisitions. The last one has the advantage of avoiding sample damage and optical aberrations when using a quartz refractive index (RI) matching solution. However, commercially available quartz cuvettes for such large samples are expensive. We propose the use of polydimethylsiloxane (PDMS) for creating tailor-made cuvettes for sample holding. For validation, we compared PDMS and quartz cuvettes by measuring light transmittance and performing whole mouse-brain imaging with LSFM. Moreover, imaging can be performed using an inexpensive RI matching solution, which further reduces the cost of the imaging process. Worth of note, the RI matching solution used in combination with PDMS leads to a moderate expansion of the sample with respect to its original size, which may represent an advantage when investigating small components, such as neuronal processes. Overall, we found the use of custom-made PDMS cuvettes advantageous in term of cost, image quality, or preservation of sample integrity with respect to other whole-mouse brain mounting strategies adopted for LSFM

Tailored Sample Mounting for Light-Sheet Fluorescence Microscopy of Clarified Specimens by Polydimethylsiloxane Casting

The combination of biological tissue clearing methods with light-sheet fluorescence microscopy (LSFM) allows acquiring images of specific biological structures of interest at whole organ scale and microscopic resolution. Differently to classical epifluorescence techniques, where the sample is cut into slices, LSFM preserves the whole organ architecture, which is of particular relevance for investigations of long-range neuronal circuits. This imaging modality comes with the need of new protocols for sample mounting. Gel matrix, hooks, tips, glues, and quartz cuvettes have been used to keep whole rodent organs in place during image acquisitions. The last one has the advantage of avoiding sample damage and optical aberrations when using a quartz refractive index (RI) matching solution. However, commercially available quartz cuvettes for such large samples are expensive. We propose the use of polydimethylsiloxane (PDMS) for creating tailor-made cuvettes for sample holding. For validation, we compared PDMS and quartz cuvettes by measuring light transmittance and performing whole mouse-brain imaging with LSFM. Moreover, imaging can be performed using an inexpensive RI matching solution, which further reduces the cost of the imaging process. Worth of note, the RI matching solution used in combination with PDMS leads to a moderate expansion of the sample with respect to its original size, which may represent an advantage when investigating small components, such as neuronal processes. Overall, we found the use of custom-made PDMS cuvettes advantageous in term of cost, image quality, or preservation of sample integrity with respect to other whole-mouse brain mounting strategies adopted for LSFM

Hard-magnetic cell microscaffolds from electroless coated 3D printed architectures

We report the application of 3D printing and wet metallization to the fabrication of magnetically driven microscaffolds for cell delivery

Biotinylated Photopolymers for 3D-Printed Unibody Lab-on-a-Chip Optical Platforms

The present work reports the first demonstration of straightforward fabrication of monolithic unibody lab-on-a-chip (ULOCs) integrating bioactive micrometric 3D scaffolds by means of multimaterial stereolithography (SL). To this end, a novel biotin-conjugated photopolymer is successfully synthesized and optimally formulated to achieve high-performance SL-printing resolution, as demonstrated by the SL-fabrication of biotinylated structures smaller than 100 µm. By optimizing a multimaterial single-run SL-based 3D-printing process, such biotinylated microstructures are incorporated within perfusion microchambers whose excellent optical transparency enables real-time optical microscopy analyses. Standard biotin-binding assays confirm the existence of biotin-heads on the surfaces of the embedded 3D microstructures and allow to demonstrate that the biofunctionality of biotin is not altered during the SL-printing, thus making it exploitable for further conjugation with other biomolecules. As a step forward, an in-line optical detection system is designed, prototyped via SL-printing and serially connected to the perfusion microchambers through customized world-to-chip connectors. Such detection system is successfully employed to optically analyze the solution flowing out of the microchambers, thus enabling indirect quantification of the concentration of target interacting biomolecules. The successful application of this novel biofunctional photopolymer as SL-material enables to greatly extend the versatility of SL to directly fabricate ULOCs with intrinsic biofunctionality

Stereolithography of perfluoropolyethers for the microfabrication of robust omniphobic surfaces

In this work, we provide a simple and straightforward method for the fabrication of stable highly hydrophobic and oleophobic surfaces by applying stereolithography (SL) to perfluoropolyethers (PFPEs). Inspired by the liquid repellency widely shown in nature, our approach enables to easily mimic the interplay between the chemistry and physics by microtexturing low surface tension PFPEs. To this end, UV-curable resins suitable for SL-processing were formulated by blending multifunctional (meth-)acrylates PFPEs oligomers with photoinitiator and visible dyes whose content was tuned to tailor resin SL sensitivities. Photocalorimetric studies were also performed to investigate the curing behavior of the different formulations upon SL light exposure. Being the first example of stereolithography applied to PFPEs, stereolithographic processability of new developed PFPEs photopolymer was compared with a standard photoresist taken as benchmark (DL260®). Optimized formulations were characterized by reduced laser penetration depth (<75 μm) and small critical energies thus enabling for fast printing of micrometric structures. Arrays of cylindrical pillars (85 μm diameter, 400 μm height) characterized by varied pillars spacing (200 ÷ 350 μm) were rapidly printed with high fidelity as attested by SEM examination. Contact angle measurements in static and dynamic conditions were performed to investigate the surface properties of textured samples using water and oil as the probing liquids. PFPEs liquid repellent performances were compared with those from DL260® textured surfaces arrayed by SL. High water contact angles coupled with low hysteresis asserted that high hydrophobic surfaces were successfully obtained and best-performing textured surfaces were also characterized by high oil repellency. Finally, this study demonstrated that omniphobic surfaces can be easily realized via a single-step, cost-effective, and time-saving process

Self-activating metal-polymer composites for the straightforward selective metallization of 3D printed parts by stereolithography

The integration of multifunctional elements directly embedded in three-dimensional (3D) printed parts is the cutting-edge of additive manufacturing (AM) and it is crucial for enlarging as well as for strengthening AM role in industrial applications. Here, a straightforward and low-cost method that synergically combines stereolithography (SLA) and selective electroless metallization (EM) is presented for the fabrication of 3D parts characterized by complex shapes and end-use multifunctionalities (conductive, magnetic, mechanical properties). To this end, a novel photocurable composite based on acrylate resin loaded with nickel (Ni) particles is developed for high-resolution SLA-printing of features with self-catalytic properties for EM. Ni particles are loaded in the resin to trigger metal deposition avoiding time consuming and expensive laser-based surface activation. The effect of Ni content on SLA behavior as well as on the efficiency of EM process is studied. Metallized SLA cured samples show good electrical and magnetic properties as well as improved robustness with respect to their non-loaded counterparts. Then, selective metallization of 3D printed parts is successfully achieved by implementing a multi-material SLA-printing where loaded and non-loaded resins are properly interchanged with strong adhesion at the interface, thus offering a cost-effective approach for rapid prototyping of functional free-form features on 3D structures

Design and Synthesis of Novel Raman Reporters for Bioorthogonal SERS Nanoprobes Engineering

Surface-enhanced Raman spectroscopy (SERS) exploiting Raman reporter-labeled nanoparticles (RR@NPs) represents a powerful tool for the improvement of optical bio-assays due to RRs&rsquo; narrow peaks, SERS high sensitivity, and potential for multiplexing. In the present work, starting from low-cost and highly available raw materials such as cysteamine and substituted benzoic acids, novel bioorthogonal RRs, characterized by strong signal (103 counts with FWHM &lt; 15 cm&minus;1) in the biological Raman-silent region (&gt;2000 cm&minus;1), RRs are synthesized by implementing a versatile, modular, and straightforward method with high yields and requiring three steps lasting 18 h, thus overcoming the limitations of current reported procedures. The resulting RRs&rsquo; chemical structure has SH-pendant groups exploited for covalent conjugation to high anisotropic gold-NPs. RR@NPs constructs work as SERS nanoprobes demonstrating high colloidal stability while retaining NPs&rsquo; physical and vibrational properties, with a limit of detection down to 60 pM. RR@NPs constructs expose carboxylic moieties for further self-assembling of biomolecules (such as antibodies), conferring tagging capabilities to the SERS nanoprobes even in heterogeneous samples, as demonstrated with in vitro experiments by transmembrane proteins tagging in cell cultures. Finally, thanks to their non-overlapping spectra, we envision and preliminary prove the possibility of exploiting RR@NPs constructs simultaneously, aiming at improving current SERS-based multiplexing bioassays

Biomimicry thinking: Methodological improvements and practical implementation

The importance of oil-water separation processes is rapidly increasing in the modern industry. This family of processes, in fact, is of fundamental importance in solving problems in several industrial sectors, among which, for instance, are the management of oil spills, accidental industrial waste water dispersions, water treatment industry, engine fuel filtration and oil sand tailing technology. This paper, firstly, presents and discusses a biologically inspired design approach, based on biomimicry thinking (BT). Besides integrating linguistic tools and brainstorming within the BT approach, the importance of a stricter integration with the engineering context is also discussed. Secondly, the paper presents a practical application of this approach: design an innovative and sustainable oil-water separation device. In particular, the application of the proposed approach allowed identifying a bioinspired solution capable to improve the process performances in a device based on a known strategy. More in detail, the adopted bioinspired solution consists of a superoleophobic surface that mimics the microstructure of the filefish scale surface and eases the oil-water separation process by hindering oil drops deposition on some surfaces. The cost and resource effectiveness of the practical realisation of this surface greatly benefited by the latest advancements in the additive manufacturing field

Fiber-Based SERS-Fluidic Polymeric Platforms for Improved Optical Analysis of Liquids

Downsizing surface-enhanced Raman spectroscopy (SERS) within microfluidic devices has opened interesting perspectives for the development of low-cost and portable (bio)sensors for the optical analysis of liquid samples. Despite the research efforts, SERS-fluidic devices still rely either on the use of expensive bulky set-ups or on polymeric devices giving spurious background signals fabricated via expensive manufacturing processes. Here, polymeric platforms integrating fluidics and optics were fabricated with versatile designs allowing easy coupling with fiber-based Raman systems. For the first time, anti-fouling photocurable perfluoropolyether (PFPE) was explored for high-throughput SERS-integrating chip fabrication via replica molding of negative stamps obtained through standard and advanced fabrication processes. The PFPE devices comprised networks of channels for fluid handling and for optical fiber housing with multiple orientations. Embedded microfeatures were used to control the relative positioning of the fibers, thus guaranteeing the highest signal delivering and collection. The feasibility of PFPE devices as fiber-based SERS fluidic platforms was demonstrated through the straightforward acquisition of Raman-SERS spectra of a mixture of gold nanoparticles as SERS substrates with rhodamine 6G (Rh6G) at decreasing concentrations. In the presence of high-performing gold nanostars, the Rh6G signal was detectable at dilutions down to the nanomolar level even without tight focusing and working at low laser power—a key aspect for analyte detection in real-world biomedical and environmental applications