In vivo label-free tissue histology through a microstructured imaging window.

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies.

Microfluidic Lab-on-a-Chip for Studies of Cell Migration under Spatial Confinement.

Understanding cell migration is a key step in unraveling many physiological phenomena and predicting several pathologies, such as cancer metastasis. In particular, confinement has been proven to be a key factor in the cellular migration strategy choice. As our insight in the field improves, new tools are needed in order to empower biologists' analysis capabilities. In this framework, microfluidic devices have been used to engineer the mechanical and spatial stimuli and to investigate cellular migration response in a more controlled way. In this work, we will review the existing technologies employed in the realization of microfluidic cellular migration assays, namely the soft lithography of PDMS and hydrogels and femtosecond laser micromachining. We will give an overview of the state of the art of these devices, focusing on the different geometrical configurations that have been exploited to study specific aspects of cellular migration. Our scope is to highlight the advantages and possibilities given by each approach and to envisage the future developments in in vitro migration studies under spatial confinement in microfluidic devices.

Rapid Prototyping of 3D Biochips for Cell Motility Studies Using Two-Photon Polymerization.

The study of cellular migration dynamics and strategies plays a relevant role in the understanding of both physiological and pathological processes. An important example could be the link between cancer cell motility and tumor evolution into metastatic stage. These strategies can be strongly influenced by the extracellular environment and the consequent mechanical constrains. In this framework, the possibility to study the behavior of single cells when subject to specific topological constraints could be an important tool in the hands of biologists. Two-photon polymerization is a sub-micrometric additive manufacturing technique that allows the fabrication of 3D structures in biocompatible resins, enabling the realization of ad hoc biochips for cell motility analyses, providing different types of mechanical stimuli. In our work, we present a new strategy for the realization of multilayer microfluidic lab-on-a-chip constructs for the study of cell motility which guarantees complete optical accessibility and the possibility to freely shape the migration area, to tailor it to the requirements of the specific cell type or experiment. The device includes a series of micro-constrictions that induce different types of mechanical stress on the cells during their migration. We show the realization of different possible geometries, in order to prove the versatility of the technique. As a proof of concept, we present the use of one of these devices for the study of the motility of murine neuronal cancer cells under high physical confinement, highlighting their peculiar migration mechanisms.

Effects of Thermal Annealing on Femtosecond Laser Micromachined Glass Surfaces.

Femtosecond laser micromachining (FLM) of fused silica allows for the realization of three-dimensional embedded optical elements and microchannels with micrometric feature size. The performances of these components are strongly affected by the machined surface quality and residual roughness. The polishing of 3D buried structures in glass was demonstrated using different thermal annealing processes, but precise control of the residual roughness obtained with this technique is still missing. In this work, we investigate how the FLM irradiation parameters affect surface roughness and we characterize the improvement of surface quality after thermal annealing. As a result, we achieved a strong roughness reduction, from an average value of 49 nm down to 19 nm. As a proof of concept, we studied the imaging performances of embedded mirrors before and after thermal polishing, showing the capacity to preserve a minimum feature size of the reflected image lower than µ5µm. These results allow for us to push forward the capabilities of this enabling fabrication technology, and they can be used as a starting point to improve the performances of more complex optical elements, such as hollow waveguides or micro-lenses.

Generation of deep ultraviolet sub-2-fs pulses.

We demonstrate the generation of few-cycle deep ultraviolet pulses via frequency upconversion of 5-fs near-infrared pulses in argon using a laser-fabricated gas cell. The measured spectrum extends from 210 to 340 nm, corresponding to a transform-limited pulse duration of 1.45 fs. We extract from a dispersion-free second-order cross-correlation measurement a pulse duration of 1.9 fs, defining a new record in the deep ultraviolet spectral range.

Editorial for the Special Issue on Ultrafast Laser Fabrication for Lab-on-a-Chip.

Ultrafast laser microfabrication is a very powerful method for producing integrated devices in transparent materials [1].[...].

Disposable Optical Stretcher Fabricated by Microinjection Moulding.

Microinjection moulding combined with the use of removable inserts is one of the most promising manufacturing processes for microfluidic devices, such as lab-on-chip, that have the potential to revolutionize the healthcare and diagnosis systems. In this work, we have designed, fabricated and tested a compact and disposable plastic optical stretcher. To produce the mould inserts, two micro manufacturing technologies have been used. Micro electro discharge machining (µEDM) was used to reproduce the inverse of the capillary tube connection characterized by elevated aspect ratio. The high accuracy of femtosecond laser micromachining (FLM) was exploited to manufacture the insert with perfectly aligned microfluidic channels and fibre slots, facilitating the final composition of the optical manipulation device. The optical stretcher operation was tested using microbeads and red blood cells solutions. The prototype presented in this work demonstrates the feasibility of this approach, which should guarantee real mass production of ready-to-use lab-on-chip devices.

Microfluidic Based Optical Microscopes on Chip.

Last decade's advancements in optofluidics allowed obtaining an ever increasing integration of different functionalities in lab on chip devices to culture, analyze, and manipulate single cells and entire biological specimens. Despite the importance of optical imaging for biological sample monitoring in microfluidics, imaging is traditionally achieved by placing microfluidics channels in standard bench-top optical microscopes. Recently, the development of either integrated optical elements or lensless imaging methods allowed optical imaging techniques to be implemented in lab on chip systems, thus increasing their automation, compactness, and portability. In this review, we discuss known solutions to implement microscopes on chip that exploit different optical methods such as bright-field, phase contrast, holographic, and fluorescence microscopy.

Rapid Prototyping of Plastic Lab-on-a-Chip by Femtosecond Laser Micromachining and Removable Insert Microinjection Molding.

We have introduced a new hybrid fabrication method for lab-on-a-chip devices through the combination of femtosecond laser micromachining and removable insert micro-injection molding. This method is particularly suited for the fast prototyping of new devices, while maintaining a competitive low cost. To demonstrate the effectiveness of our approach, we designed, fabricated, and tested a completely integrated flow cytometer coupled to a portable media device. The system operation was tested with fluorescent plastic micro-bead solutions ranging from 100 beads/µL to 500 beads/µL. We demonstrated that this hybrid lab-on-a-chip fabrication technology is suitable for producing low-cost and portable biological microsystems and for effectively bridging the gap between new device concepts and their mass production.

Femtosecond laser microstructuring for polymeric lab-on-chips.

This paper provides an overview of femtosecond laser microfabrication in polymeric materials, with emphasis on lab-on-chip applications. Due to the nonlinear interaction of femtosecond laser pulses with polymers, laser-induced modifications are localized to the focal volume, enabling high resolution patterning in 3D. Femtosecond laser microfabrication offers unmatched versatility in fabricating surface microchannels and diffractive optics by means of laser ablation, buried optical waveguides and micro-optics through refractive index modification and complex 3D microstructures in photoresists by two-photon polymerization. Femtosecond laser microfabrication technology opens the door to fabricating integrated lab-on-chip devices with a single tool.

Optical sensing in microfluidic lab-on-a-chip by femtosecond-laser-written waveguides.

We use direct femtosecond laser writing to integrate optical waveguides into a commercial fused silica capillary electrophoresis chip. High-quality waveguides crossing the microfluidic channels are fabricated and used to optically address, with high spatial selectivity, their content. Fluorescence from the optically excited volume is efficiently collected at a 90 degree angle by a high numerical aperture fiber, resulting in a highly compact and portable device. To test the platform we performed electrophoresis and detection of a 23-mer oligonucleotide plug. Our approach is quite powerful because it allows the integration of photonic functionalities, by simple post-processing, into commercial LOCs fabricated with standard techniques.

Fluorescence monitoring of microchip capillary electrophoresis separation with monolithically integrated waveguides.

Using femtosecond laser writing, optical waveguides were monolithically integrated into a commercial microfluidic lab-on-a-chip device, with the waveguides intersecting a microfluidic channel. Continuous-wave laser excitation through these optical waveguides confines the excitation window to a width of 12 microm, enabling high-resolution monitoring of the passage of different types of fluorescent analytes when migrating and being separated in the microfluidic channel by microchip capillary electrophoresis. Furthermore, we demonstrate on-chip-integrated waveguide excitation and detection of a biologically relevant species, fluorescently labeled DNA molecules, during microchip capillary electrophoresis. Well-controlled plug formation as required for on-chip integrated capillary electrophoresis separation of DNA molecules, and the combination of waveguide excitation and a low limit of detection, will enable monitoring of extremely small quantities with high spatial resolution.