GC and MAGO acknowledge all members of the CeMi. MSS acknowledges support via an EPSRC Programme Grant (EP/P001114/1).
GC: software (contribution to software development and algorithms), formal analysis (analysis of nanoindentation data), validation, Investigation (nanoindentation experiments on polyacrylamide gels), data curation, writing (original draft, review and editing), visualization (figures and graphs). MAGO: investigation (preparation of gels and cells samples, nanoindentation experiments on cells), writing (original draft, review and editing), visualization (figures and graphs). NA: validation, writing (review and editing). IL: software (contribution to software development and algorithms), validation, writing (review and editing); MV: conceptualization, software (design and development of original software and algorithms), validation, resources, writing (original draft, review and editing), supervision, project administration, funding acquisition MSS: resources, writing (review and editing), supervision, project administration, funding acquisition. All authors read and approved the final manuscript.&#160;&#160;

The authors have nothing to disclose.

This protocol shows how to robustly acquire force spectroscopy nanoindentation data using a commercially available ferrule-top nanoindenter on both hydrogels and single cells. In addition, instructions for the use of an open-source software programmed in Python comprising a precise workflow for the analysis of nanoindentation data are provided.
Critical steps in the protocol 
The following steps have been identified to be of particular importance when following this proto...

The fundamental role of mechanics in biology is nowadays established1,2. From whole tissues to single cells, mechanical properties can inform about the pathophysiological state of the biomaterial under investigation3,4. For example, breast tissue affected by cancer is stiffer than healthy tissue, a concept that is the basis of the popular palpation test5. Notably, it has been recently shown that the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is underlined by changes in the mechanical properties of blood cells, including decreased erythrocyte deformability and decreased lymphocyte and neutrophil stiffness as compared to blood cells from SARS-CoV-2-na&#239;ve individuals6.
In general, the mechanics of cells and tissues are inherently intertwined: each tissue has specific mechanical properties that simultaneously influence and depend on those of the constituent cells and extracellular matrix (ECM)5. Because of this, strategies to study mechanics in biology often involve engineering substrates with physiologically relevant mechanical stimuli to elucidate cell behavior in response to those stimuli. For example, the seminal work by Engler and colleagues demonstrated that mesenchymal stem cell linage commitment is controlled by matrix elasticity, as studied on soft and stiff two-dimensional polyacrylamide (PAAm) hydrogels7.
Many strategies to mechanically characterize the biomaterial under investigation exist, varying in spatial scale (i.e., local to bulk) and in the mode of deformation (e.g., axial vs shear), consequently yielding different information, which needs careful interpretation3,8,9,10. The mechanics of soft biomaterials is commonly expressed in terms of stiffness. However, stiffness depends on both material properties and geometry, whereas elastic moduli are fundamental properties of a material and are independent of the material&#39;s geometry11. As such, different elastic moduli are related to the stiffness of a given sample, and each elastic modulus encompasses the material&#39;s resistance to a specific mode of deformation (e.g., axial vs shear) under different boundary conditions (e.g., free expansion vs confinement)11,12. Nanoindentation experiments allow the quantification of mechanical properties through the E which is associated with uniaxial deformation (indentation) when the biomaterial is not laterally confined10,11,12.
The most popular method to quantify&#160;E of biological systems at the microscale is AFM13,14,15,16. AFM is an extremely powerful tool with force resolution down to the pN&#160;level and spatial resolution down to the sub-nm scale. Further, AFM offers extreme flexibility in terms of coupling with complementary optical and mechanical tools, extending its capabilities to extract a wealth of information from the biomaterial under investigation13. Those attractive features, however, come with a barrier-to-entry represented by the complexity of the experimental set-up. AFM requires extensive training before users can acquire robust data, and its use for everyday mechanical characterization of biological materials is often unjustified, especially when its unique force and spatial resolutions are not required.
Because of this, a new class of nanoindenters has recently gained popularity due to their ease of use, while still offering AFM-comparable data with sub-nN force resolution and &#181;m spatial resolution, reflecting forces exerted and perceived by cells over relevant length scales2. Particularly, ferrule-top nanoindentation devices based on optical fiber sensing technology17,18 have gained popularity among researchers active in the field of mechanobiology and beyond; and a wealth of works reporting the mechanical properties of biomaterials using these devices, including cells19,20, hydrogels8,21, and tissues22,23 have been published. Despite the capabilities of these systems to probe local dynamic mechanical properties (i.e., storage and loss modulus), quasi-static experiments yielding E remain the most popular choice8,19,20,21. In brief, quasi-static nanoindentation experiments&#160;consists of indenting the sample with a constant speed up to a set-point defined either by a maximum displacement, force, or indentation depth, and recording both the force and the vertical position of the cantilever in so-called force-distance (F-z) curves. F-z curves are then converted into force-indentation (F-&#948;) curves through the identification of the contact point (CP), and fitted with an appropriate contact mechanics model (usually the Hertz model13) to compute E.
While the operation of ferrule-top nanoindenters resembles AFM measurements, there are specificities worth considering. In this work, a step-by-step guide to robustly acquire F-z curves from cells and tissue-mimicking hydrogels using a commercially available ferrule-top nanoindenter is provided, in order to encourage standardization of experimental procedures between research groups using this and other similar devices. In addition, advice on how to best prepare hydrogel samples and cells to perform nanoindentation experiments is given, together with troubleshooting tips along the experimental pathway.
Furthermore, much of the variability in nanoindentation results (i.e., E and its distribution) depends on the specific procedure used to analyze data, which is non-trivial. To address this issue, instructions for the use of a newly developed open-source software programmed in Python and equipped with a user-friendly graphical user interface (GUI) for batch analysis of F-z curves are provided. The software allows for fast data screening, filtering of data, computation of the CP through different numerical procedures, the conventional computation of E, as well a more advanced analysis named the elasticity spectra24, allowing to estimate the cell&#39;s bulk Young&#39;s modulus, actin cortex&#39;s Young&#39;s modulus, and actin cortex&#39;s thickness. The software can be freely downloaded from GitHub and can be easily adapted to analyze data originating from other systems by adding an appropriate data parser. It is emphasized that this protocol can be used for other ferrule-top nanoindentation devices, and other nanoindentation devices in general, granted some steps are adapted according to the specific instrument&#39;s guidelines. The protocol is schematically summarized in Figure 1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12 mm coverslips</td>
			<td>VWR</td>
			<td>631-1577P</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm cell treated culture dishes</td>
			<td>Greiner CELLSTAR</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A4058</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylsilane</td>
			<td>Alfa Aesar</td>
			<td>L16400</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Merk</td>
			<td>7727-54-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisacrylamide</td>
			<td>Merk</td>
			<td>110-26-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Chiaro nanoindenter</td>
			<td>Optics 11 Life</td>
			<td></td>
			<td>&#160;no catologue number</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>general</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>High glucose DMEM</td>
			<td>Gibco</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td></td>
			<td></td>
			<td>general</td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimberly Clark</td>
			<td>21905-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope glass slides</td>
			<td>VWR</td>
			<td>631-1550P</td>
			<td></td>
		</tr>
		<tr>
			<td>MilliQ system</td>
			<td>Merk Millipore</td>
			<td>ZR0Q008WW</td>
			<td></td>
		</tr>
		<tr>
			<td>OP1550 Interferometer</td>
			<td>Optics11 Life</td>
			<td></td>
			<td>no catalogue number</td>
		</tr>
		<tr>
			<td>Optics 11 Life probe (k = 0.02-0.005 N/m, R = 3-3.5 um)</td>
			<td>Optics 11 Life</td>
			<td></td>
			<td>no catologue number</td>
		</tr>
		<tr>
			<td>Optics 11 Life probe (k = 0.46-0.5 N/m, R = 50-55 um)</td>
			<td>Optics 11 Life</td>
			<td></td>
			<td>no catologue number</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>RainX rain repellent</td>
			<td>RainX</td>
			<td>26012</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard petri dishes (90 mm)</td>
			<td>Thermo Scientific</td>
			<td>101RTIRR</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>110-18-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum dessicator</td>
			<td>Thermo Scientific</td>
			<td>531-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition software (v 3.4.1)</td>
			<td>Optics 11 Life</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GitHub Desktop (Optional)</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Python 3</td>
			<td>Python Software Foundation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visual Studio Code (Optional)</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

experimental and data analysis workflow for soft matter nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

Following the protocol, a set of F-z curves is obtained. The dataset will most likely contain good curves, and curves to be discarded before continuing with the analysis. In general, curves should be discarded if their shape is different from the one shown in Figure 4A. Figure 5AI shows a dataset of ~100 curves obtained on a soft PAAm hydrogel of expected E 0.8 KPa35 uploaded in the NanoPrepare GUI. Most curves present a...

Watch this Scientific Journal Video about Experimental and Data Analysis Workflow for Soft Matter Nanoindentation at JoVE.com

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

experimental-data-analysis-workflow-for-soft-matter

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3.6K Views.  University of Glasgow. The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

Video: Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Soft Lithographic Functionalization and Patterning Oxide-free Silicon and Germanium

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. 
Microcontact printing (&mu;CP) is a soft-lithographic technique for patterning SAMs on myriad surfaces.1-9 Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 &mu;m.10-16
In contrast to traditional printing, inkless &mu;CP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (&lt;200 nm) features.17-23 However, up till now, inkless &mu;CP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation.
Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many &mu;CP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

Here we describe a simple method for patterning oxide-free silicon and germanium with reactive organic monolayers and demonstrate functionalization of the patterned substrates with small molecules and proteins. The approach completely protects surfaces from chemical oxidation, provides precise control over feature morphology, and provides ready access to chemically discriminated patterns.

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Studying Soft-matter and Biological Systems over a Wide Length-scale from Nanometer and Micrometer Sizes at the Small-angle Neutron Diffractometer KWS-2

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. The instrument is optimized for the exploration of the wide momentum transfer Q range between 1x10-4 and 0.5 &#197;-1 by combining classical pinhole, focusing (with lenses), and time-of-flight (with chopper) methods, while simultaneously providing high-neutron intensities with an adjustable resolution. Because of its ability to adjust the intensity and the resolution within wide limits during the experiment, combined with the possibility to equip specific sample environments and ancillary devices, the KWS-2 shows a high versatility in addressing the broad range of structural and morphological studies in the field. Equilibrium structures can be studied in static measurements, while dynamic and kinetic processes can be investigated over time scales between minutes to tens of milliseconds with time-resolved approaches. Typical systems that are investigated with the KWS-2 cover the range from complex, hierarchical systems that exhibit multiple structural levels (e.g., gels, networks, or macro-aggregates) to small and poorly-scattering systems (e.g., single polymers or proteins in solution). The recent upgrade of the detection system, which enables the detection of count rates in the MHz range, opens new opportunities to study even very small biological morphologies in buffer solution with weak scattering signals close to the buffer scattering level at high Q.
In this paper, we provide a protocol to investigate samples with characteristic size levels spanning a wide length scale and exhibiting ordering in the mesoscale structure using KWS-2. We present in detail how to use the multiple working modes that are offered by the instrument and the level of performance that is achieved.

Here, we present a protocol to investigate soft matter and biophysical systems over a wide mesoscopic length scale, from nm to &#181;m that involves the use of the KWS-2 SANS diffractometer at high intensities and an adjustable resolution.

The KWS-2 SANS diffractometer is dedicated to the investigation of soft matter and biophysical systems covering a wide length scale, from nm to &#181;m. ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Bioinspired Soft Robot with Incorporated Microelectrodes

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics paradigm, especially in biomedical research. Recreating artificial life-like actuation dynamics is crucial for a soft-robotic system. However, the precise control and tuning of actuation behavior still represents one of the main challenges of modern soft robotic systems. This method describes a low-cost, highly scalable, and easy-to-use procedure to fabricate an electrically controllable soft robot with life-like movements that is activated and controlled by the contraction of cardiac muscle tissue on a micropatterned sting ray-like hydrogel scaffold. The use of soft photolithography methods makes it possible to successfully integrate multiple components in the soft robotic system, including micropatterned hydrogel-based scaffolds with carbon nanotubes (CNTs) embedded gelatin methacryloyl (CNT-GelMA), poly(ethylene glycol) diacrylate (PEGDA), flexible gold (Au) microelectrodes, and cardiac muscle tissue. In particular, the hydrogels alignment and micropattern are designed to mimic the muscle and cartilage structure of the sting ray. The electrically conductive CNT-GelMA hydrogel acts as a cell scaffold that improves the maturation and contraction behavior of cardiomyocytes, while the mechanically robust PEGDA hydrogel provides structural cartilage-like support to the whole soft robot. To overcome the hard and brittle nature of metal-based microelectrodes, we designed a serpentine pattern that has high flexibility and can avoid hampering the beating dynamics of cardiomyocytes. The incorporated flexible Au microelectrodes provide electrical stimulation across the soft robot, making it easier to control the contraction behavior of cardiac tissue.

A bioinspired scaffold is fabricated by a soft photolithography technique using mechanically robust and electrically conductive hydrogels. The micropatterned hydrogels provide directional cardiomyocyte cell alignment, resulting in a tailored direction of actuation. Flexible microelectrodes are also integrated into the scaffold to bring electrical controllability for a self-actuating cardiac tissue.

Bioinspired soft robotic systems that mimic living organisms using engineered muscle tissue and biomaterials are revolutionizing the current biorobotics ...

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

We present a freely available workflow built for rapid exploration and accurate analysis of cellular bodies in specific cell compartments in fluorescence microscopy images. This user-friendly workflow is designed on the open-source software Icy and also uses ImageJ functionalities. The pipeline is affordable without knowledge in image analysis.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in ...