Infrared imaging with visible light in microfluidic devices: the water absorption barrier.

Infrared spectro-microscopy is a powerful technique for analysing chemical maps of cells and tissues for biomedical and clinical applications, yet the strong water absorption in the mid-infrared region is a challenge to overcome, as it overlaps with the spectral fingerprints of biological components. Microfluidic chips offer ultimate control over the water layer thickness and are increasingly used in infrared spectro-microscopy. However, the actual impact of the water layer thickness on the instrument's performance is often left to the experimentalist's intuition and the peculiarities of specific instruments.

Ca-dependent vesicular and non-vesicular lipid transfer controls hypoosmotic plasma membrane expansion.

Robust coordination of surface and volume changes is critical for cell integrity. Few studies have elucidated the plasma membrane (PM) remodeling events during cell surface and volume alteration, especially regarding PM sensing and its subsequent rearrangements. Here, using fission yeast protoplasts, we reveal a Ca-dependent mechanism for membrane addition that ensures PM integrity and allows its expansion during acute hypoosmotic cell swelling.

3D Compartmentalised Human Pluripotent Stem Cell-derived Neuromuscular Co-cultures.

Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co-culture of neural spheroids composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro.

Photoreceptor laminin drives differentiation of human pluripotent stem cells to photoreceptor progenitors that partially restore retina function.

Blindness caused by advanced stages of inherited retinal diseases and age-related macular degeneration are characterized by photoreceptor loss. Cell therapy involving replacement with functional photoreceptor-like cells generated from human pluripotent stem cells holds great promise. Here, we generated a human recombinant retina-specific laminin isoform, LN523, and demonstrated the role in promoting the differentiation of human embryonic stem cells into photoreceptor progenitors.

Bioengineering a miniaturized in vitro 3D myotube contraction monitoring chip to model muscular dystrophies.

Quantification of skeletal muscle functional contraction is essential to assess the outcomes of therapeutic procedures for neuromuscular disorders. Muscle three-dimensional "Organ-on-chip" models usually require a substantial amount of biological material, which rarely can be obtained from patient biopsies. Here, we developed a miniaturized 3D myotube culture chip with contraction monitoring capacity at the single cell level.

A High-Throughput Platform for Culture and 3D Imaging of Organoids.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.

Automated high-speed 3D imaging of organoid cultures with multi-scale phenotypic quantification.

Current imaging approaches limit the ability to perform multi-scale characterization of three-dimensional (3D) organotypic cultures (organoids) in large numbers. Here, we present an automated multi-scale 3D imaging platform synergizing high-density organoid cultures with rapid and live 3D single-objective light-sheet imaging. It is composed of disposable microfabricated organoid culture chips, termed JeWells, with embedded optical components and a laser beam-steering unit coupled to a commercial inverted microscope.

Integration of a microfluidic multicellular coculture array with machine learning analysis to predict adverse cutaneous drug reactions.

Adverse cutaneous reactions are potentially life-threatening skin side effects caused by drugs administered into the human body. The availability of a human-specific platform that can prospectively screen drugs and predict this risk is therefore of great importance to drug safety. However, since adverse cutaneous drug reactions are mediated by at least 2 distinct mechanisms, both involving systemic interactions between liver, immune and dermal tissues, existing skin models have not been able to comprehensively recapitulate these complex, multi-cellular interactions to predict the skin-sensitization potential of drugs.

Hyperspectral infrared microscopy with visible light.

Hyperspectral microscopy is an imaging technique that provides spectroscopic information with high spatial resolution. When applied in the relevant wavelength region, such as in the infrared (IR), it can reveal a rich spectral fingerprint across different regions of a sample. Challenges associated with low efficiency and high cost of IR light sources and detector arrays have limited its broad adoption.

Fatty acids as biomimetic replication agents for luminescent metal-organic framework patterns.

Luminescent metal-organic frameworks (MOFs) are known to spontaneously self-assemble on human fingerprints. Here, we investigate the different chemical components of fingerprints and determine that MOF growth is predominantly induced by insoluble fatty acids. This finding shows that these simple biomolecules can be employed for the precise positioning of luminescent MOFs.

High pulsed power VCSEL arrays with polymer microlenses formed by photoacid diffusion.

We demonstrate millimeters-long VCSEL linear arrays with SU-8 epoxy-based microlenses that are directly patterned and cross-linked on the output apertures by a simple, photoacid-diffusion-aided photolithography technique. The linear arrays are capable of delivering >7 W of peak pulsed output power. By exploiting the photoacid diffusion effect, it is possible to produce a range of microlens structures with height and radius of curvature ranging from approximately ten to tens of microns.

Cell response to substrate rigidity is regulated by active and passive cytoskeletal stress.

Morphogenesis, tumor formation, and wound healing are regulated by tissue rigidity. Focal adhesion behavior is locally regulated by stiffness; however, how cells globally adapt, detect, and respond to rigidity remains unknown. Here, we studied the interplay between the rheological properties of the cytoskeleton and matrix rigidity.

Forces and constraints controlling podosome assembly and disassembly.

Podosomes are a singular category of integrin-mediated adhesions important in the processes of cell migration, matrix degradation and cancer cell invasion. Despite a wealth of biochemical studies, the effects of mechanical forces on podosome integrity and dynamics are poorly understood. Here, we show that podosomes are highly sensitive to two groups of physical factors.

Circulating Tumor Cell cluster phenotype allows monitoring response to treatment and predicts survival.

Circulating tumor cells (CTCs) are putative markers of tumor prognosis and may serve to evaluate patient's response to chemotherapy. CTCs are often detected as single cells but infrequently as clusters and are indicative of worse prognosis. In this study, we developed a short-term culture of nucleated blood cells which was applied to blood samples from breast, lung, esophageal and bladder cancer patients.

Integrating Microfabrication into Biological Investigations: the Benefits of Interdisciplinarity.

The advent of micro and nanotechnologies, such as microfabrication, have impacted scientific research and contributed to meaningful real-world applications, to a degree seen during historic technological revolutions. Some key areas benefitting from the invention and advancement of microfabrication platforms are those of biological and biomedical sciences. Modern therapeutic approaches, involving point-of-care, precision or personalized medicine, are transitioning from the experimental phase to becoming the standard of care.

Low-dose anti-inflammatory combinatorial therapy reduced cancer stem cell formation in patient-derived preclinical models for tumour relapse prevention.

Background: Emergence of drug-resistant cancer phenotypes is a challenge for anti-cancer therapy. Cancer stem cells are identified as one of the ways by which chemoresistance develops.

Method: We investigated the anti-inflammatory combinatorial treatment (DA) of doxorubicin and aspirin using a preclinical microfluidic model on cancer cell lines and patient-derived circulating tumour cell clusters.

Artificial Microniche Array with Spatially Structured Biochemical Cues.

We present here a method to create arrays of microcavities that can be differentially coated on their bottom, side, and top with different proteins. These cavities range in size from single cell to multicellular aggregate. We provide detailed protocols to create such arrays with some variations using different materials and different coating proteins.

Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device.

The development of personalized cancer therapy depends on a robust system to monitor the patient's individual response to anticancer treatment. Anticancer drug efficacy has been tested on circulating tumor cells (CTCs) derived from patient blood samples after ex vivo expansion into CTC clusters. Current attempts to culture these primary cancer cells focus on long-term maintenance under growth factor supplements into cell lines, which usually takes >6 months and results in a CTC expansion efficiency of <20%.

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light.

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device.

3D Protein Dynamics in the Cell Nucleus.

The three-dimensional (3D) architecture of the cell nucleus plays an important role in protein dynamics and in regulating gene expression. However, protein dynamics within the 3D nucleus are poorly understood. Here, we present, to our knowledge, a novel combination of 1) single-objective based light-sheet microscopy, 2) photoconvertible proteins, and 3) fluorescence correlation microscopy, to quantitatively measure 3D protein dynamics in the nucleus.

Liquid biopsy and therapeutic response: Circulating tumor cell cultures for evaluation of anticancer treatment.

The lack of a robust anticancer drug screening system to monitor patients during treatment delays realization of personalized treatment. We demonstrate an efficient approach to evaluate drug response using patient-derived circulating tumor cell (CTC) cultures obtained from liquid biopsy. Custom microfabricated tapered microwells were integrated with microfluidics to allow robust formation of CTC clusters without pre-enrichment and subsequent drug screening in situ.

IR-Live: fabrication of a low-cost plastic microfluidic device for infrared spectromicroscopy of living cells.

Water is a strong mid-infrared absorber, which has hindered the full exploitation of label-free and non-invasive infrared (IR) spectromicroscopy techniques for the study of living biological samples. To overcome this barrier, many researchers have built sophisticated fluidic chambers or microfluidic chips wherein the depth of the liquid medium in the sample compartment is limited to 10 μm or less. Here we report an innovative and simple way to fabricate plastic devices with infrared transparent view-ports enabling infrared spectromicroscopy of living biological samples; therefore the device is named "IR-Live".

Chemical vapour deposition of zeolitic imidazolate framework thin films.

Integrating metal-organic frameworks (MOFs) in microelectronics has disruptive potential because of the unique properties of these microporous crystalline materials. Suitable film deposition methods are crucial to leverage MOFs in this field. Conventional solvent-based procedures, typically adapted from powder preparation routes, are incompatible with nanofabrication because of corrosion and contamination risks.

3D high- and super-resolution imaging using single-objective SPIM.

Single-objective selective-plane illumination microscopy (soSPIM) is achieved with micromirrored cavities combined with a laser beam-steering unit installed on a standard inverted microscope. The illumination and detection are done through the same objective. soSPIM can be used with standard sample preparations and features high background rejection and efficient photon collection, allowing for 3D single-molecule-based super-resolution imaging of whole cells or cell aggregates.