Name: assessing collagen and elastin pressure-dependent microarchitectures in live, human resistance arteries by label-free fluorescence microscopy
Uploaded: 2018-04-09T14:45:00
Description: Watch this Scientific Journal Video about Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy at JoVE.com

The authors thank the Danish Molecular Biomedical Imaging Center at the Faculty of Natural Sciences, University of Southern Denmark, for the use of laboratories and microscopes. Kristoffer Rosenstand and Ulla Melchior are acknowledged for excellent technical assistance with the pressure myography and imaging.

Collection of biopsies of the human parietal pericardium for use in this work was performed after written informed consent, as previously described33. The study of human tissues conform to the principles outlined in the Declaration of Helsinki34 and was approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20100044 and S-20140202) and the Danish Data Protection Agency.
1. Collect Tissue and Isolate (Human) Resistance...

This work represents our suggestion for a standardized, combined imaging and pressure myography approach, valuable for simultaneous assessment of the mechanical properties of resistance arteries and pressure-related changes in the structure of the arterial wall over a pressure range from 0 to 100 mmHg. The presented approach was developed using custom built equipment, however, any pressure myograph that fits on a two-photon excitation fluorescence microscope can be used, when the design of both equipments allows imaging ...

The pathogenic contribution and effects of resistance artery remodeling are documented in essential hypertension, diabetes and the metabolic syndrome1,2,3,4,5. Deciphering the relationship between the mechanical and microarchitectural properties of the microvascular wall is essential for developing mathematical models of this association. Such models will improve understanding the remodeling process and will support the development of in silico models useful for testing pharmacological strategies targeting disease related remodeling of the arterial wall.
Prior studies focused in understanding how the microarchitecture of the arterial wall relates to arterial wall mechanics by incorporating mechanical measures and the microarchitecture of the extracellular matrix (ECM) are almost exclusively performed on large, elastic conduit arteries from mice or swine6,7,8,9,10,11. Imaging of the microstructures of the wall is typically performed using nonlinear optical techniques, taking advantage of the autofluorescence of elastin and second harmonic generation by collagen. This allows spatiotemporal imaging of the two major components of the extracellular matrix, elastin and collagen, without a need for staining. Imaging of the arterial wall in full thickness is a challenge in large conduit arteries due to scatter of the light in the thick tunica media. However, to determine how the microarchitecture of the structural components of the arterial wall relate to the observed mechanical properties, three-dimensional information must be obtained during the mechanical testing. For large arteries like the human aorta, this requires biaxial mounting, mechanical testing and imaging of regions of interest in 1-2 cm2 pieces of the arterial wall7,9,10,12. Only part of the wall can be imaged and mechanically tested.
For smaller arteries of any species (e.g., human pericardial13, pulmonary14 and subcutaneous15 arteries, rat mesenteric arteries16,17,18,19,20, mouse cremaster, mesenteric, cerebral, femoral and carotid arteries21,22,23,24,25,26,27) imaging of the entire wall thickness is possible and can be combined with mechanical testing. This allows simultaneous recording of the mechanical properties and the structural arrangements within the wall. However, a direct mathematical modeling of the relationship between the observed alterations in the three-dimensional structure of the ECM and changed mechanical properties of the resistance arterial wall, has to the best of our knowledge only been reported upon recently in human resistance arteries13,15.
In this work, an ex vivo method for passive mechanical testing and simultaneous three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries is described. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for obtaining measures of internal elastic lamina branching angles and adventitial collagen straightness13 using Fiji28. Collagen and elastin volume densities are determined using Ilastik software29 and finally, the inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed.
The goal of describing the imaging and image analyses techniques in combination with mathematical modeling is to provide investigators a systematical approach to describe and understand observed pressure induced changes in the ECM of resistance arteries. The described method is focused in quantifying the changes in the ECM in a vessel during pressurization, by comparing the structure of the ECM at 20, 40 and 100 mmHg. These pressures were chosen for determining the structure of the arterial wall at its more compliant (20 mmHg), stiff (100 mmHg) and intermediate (40 mmHg) state, respectively. However, any process in the vascular wall of live arteries, including changes induced by vasoactive components, hysteresis and flow, can be quantified, depending on the research hypothesis in question by the investigator.
The use of two-photon excitation fluorescence microscopy (TPEM) in combination with a pressure myograph for studying pressure (or other) induced changes in the ECM of live arteries is emphasized. First, because this allows simultaneous acquisition of the overall three-dimensional structure of the arterial wall (diameter and wall thickness) along with three-dimensional label-free acquisition of high quality, detailed images of the collagen and elastin microarchitectures as described13 by taking advantage of the elastin autofluorescence and the collagen second harmonic generation signal (SHG)30. Second, TPEM allows use of low-energy near-infrared excitation light, minimizing photodamage of the tissue and thus, repeated imaging at exactly the same position within the vascular wall is allowed, permitting repeated-measurements analyses of observed changes.
The use of an alternative approach using confocal imaging of pressure fixed arteries is discussed to allow users without access to TPEM an opportunity to use the described method as well. Information on ECM structure and volume densities can also be retrieved from two-dimensional analyses of tissues sectioned in serial, e.g. as described by31,32. However, due to the lack of possibility to retrieve three-dimensional structural information over the length scales of the artery as well as during changing conditions using this method, it is not recommend using this approach for investigations of pressure and treatment induced three-dimensional changes in the ECM.
The minimum requirement for the investigator to apply the herein described method is the access to a setup for cannulation and pressurization of arteries in combination with a confocal or two-photon excitation fluorescence microscope. The setup described in the following protocol is a custom-built pressure myograph with a longitudinal force transducer, built to fit on a custom built inverted two-photon excitation fluorescence microscope.

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			<td height="20" style="height:20px;width:236px;">AHF analysentechnik AG (T&#252;bingen, Germany).</td>
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			<td height="20" style="height:20px;width:236px;">AHF analysentechnik AG (T&#252;bingen, Germany).</td>
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assessing collagen and elastin pressure-dependent microarchitectures in live, human resistance arteries by label-free fluorescence microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

The custom-built pressure myograph for imaging used in this work is shown in Figure 1. Special attention for the design of the myograph was paid to i) the chamber with a small volume (2 mL) and ii) the possibility for positioning the cannulae close to, and parallel with the glass bottom (Figure 1B). The bottom of the chamber fits a 50 &#215; 24 mm #1.5 glass coverslip (replaceable). The pressure controller was built from a standa...

Watch this Scientific Journal Video about Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy at JoVE.com

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

The overall goal of this methodology is to provide a standardized procedure for obtaining images of live human resistance arteries for analysis of the relationship between the microarchitecture of the extracellular matrix and the mechanics of the arterial wall. This method can help answer key questions in vascular biology and mechanics such as how pressure induces changes in the overall geometry as well as the microarchitecture of the vascular wall. The main advantage of the technique is that you don't have to label your sample for the imaging.Though this method can provide insight into resistance artery mechanics, a modified version of the protocol can be applied to other tube-like organs like the bronchi or to strips from human bladder, or the intestinal wall. To begin, collect tissue samples of interest immediately upon their excision during surgery and prepare the vessels as described in the accompanying text protocol. Pressurize a cannulated artery to five millimeters mercury.Then set up the optical path for the imaging by activating the two-photon laser and setting the excitation wavelength to 820 nanometers. Insert a 460 nanometer long pass dichroic mirror to split the emission light between the photo multipliers and collect the emission simultaneously in two channels. Use 30 to 60 nanometer wide bandpass filters centered at 520 nanometers to collect the elastin autofluorescence and another centered at 410 nanometers to collect the collagen second harmonic generation signal.Now set the laser dwell time and pixel to 10 microseconds. Set the resolution to 512 by 512 pixels for 100%field of view. Enable continuous scanning and use as low excitation light power and pixel dwell time as possible to avoid photo damaging the live artery.Then manually scan through the artery to find the plane with the maximum diameter. Next, choose a rectangular slice covering the artery's widest diameter and scan a single frame with a pixel size of 300 nanometers per pixel or less. Save this image for analysis at a later time.To determine lumen diameter and wall thickness at the maximum diameter of the artery, first load the image in Fiji. Set the scale by clicking on main menu and then go to analyze followed by set scale. Here enter the micrometer per pixel and pixel ratio.Next, choose the Fiji line tool and draw a line between the two internal elastic lamina at each side of the arterial lumen. Then click Control M to report the length in the results table. Continue to draw more lines to measure the thickness of each wall and save the final measurement.Change to a 60X objective with a numerical aperture of one or greater. Image the collagen and elastin microarchitecture at five millimeters mercury by scanning the entire thickness of the arterial wall. Next, define the Z stack start and end depth and the Z step spacing and pixel density.When you're designing your Z stacks, make sure your region of interest is small enough to avoid any inference of curvature on your subsequent analysis. When complete, set each channel to be saved separately. Lastly, find one to three different regions of interest and obtain 3D image stacks of good quality.If your elastin autofluorescence is bleaching, you are using too high excitation light power. This should be avoided. In order to finish your ongoing experiment, you may add a dilute concentration of eosin to enhance the elastin autofluorescence.When finished, repeat the entire process at pressures varying up to 100 millimeters mercury, replacing the buffer in the myograph chamber with fresh 37 degrees Celsius HBS after each pressure step. To begin image analysis, open the elastin image stack in Fiji. Then go to image, click on stacks, and select Z project to choose the images to work with and choose max intensity for projection type.Save the max intensity projection as a TIFF image. Then choose as many internal elastic lamina fiber branching points as possible and measure them using the angle tool. Systematically work through each image and when finished, save the Fiji results sheet.To measure the waviness of the adventitial collagen, open the collagen image stack in Fiji. Next, go to image down to stacks and select Z project to choose images to work with and to set the max intensity projection. Now change the image type to an eight-bit TIFF image by clicking image, type, and then eight-bit.Save the max intensity projection as a TIFF image. Open the Neuron J plugin by clicking on Fiji and selecting Neuron J under plugins. Then open the eight-bit TIFF file in Neuron J.Once open, click on add tracings and select fibers to analyze by clicking on the start and the end of each fiber.Next, go to measure tracings and choose display tracing and vertex measurements in the dialogue box. To calculate collage fiber straightness, first copy the values in the Fiji tracings window to Excel. The length values in the tracings window are measures of the full lengths of the analyzed collagen fibers Lf.Second, copy for each collagen fiber bundle analyzed the first and last XY coordinates of the Fiji vertices window to Excel.Then calculate the length of the straight line connecting each end of the analyzed collagen fiber L0 using Pythagoras Theorem. Collagen fiber straightness is finally calculated as L0/Lf. Following the protocol just shown, the widest part of the artery was identified and at this point the artery was scanned using label-free fluorescence microscopy to measure the maximum vessel diameter and vessel wall thickness.Utilizing a higher magnification to high numerical aperture objective, not only can the vessel diameter and wall thickness be measured, but also the microarchitectures of collagen and elastin. The microarchitectures of elastin fiber branching angles and collagen straightness can be determined in consecutive image sections using the appropriate image analysis software. This allows a direct analysis of the relationship between the pressure induced changes in the microarchitectures of collagen and elastin and the incremental elastic modulus at different pressures.Following this procedure, other information can be extracted from the images as well. This could be the orientation angels of adventitial collagen along the vessel longitudinal axis or it could be the microarchitecture of the extracellular matrix in tunica media. The quantitative measures extracted from the images using this method can be fed into mathematical models in order to elucidate how the healthy and deceased human circulation respond to different conditions.After watching this video, you should have a thorough understanding on how to design your imaging experiments and subsequent image analysis in order to extract information on how pressure induces changes in the microarchitecture of the resistance artery wall. Remember that you have to avoid any exposure to visible and invisible light when working with two-photon excitation fluorescence microscopy.

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Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma membrane1. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex2,3. Traditional assays examining the interaction between channels and regulatory proteins require exogenous labels that can potentially alter the protein&#39;s behavior and decrease the physiological relevance of the target, while providing little information on the time course of interactions in living cells. Optical biosensors, such as the X-BODY Biosciences BIND Scanner system, use a novel label-free technology, resonance wavelength grating (RWG) optical biosensors, to detect changes in resonant reflected light near the biosensor. This assay allows the detection of the relative change in mass within the bottom portion of living cells adherent to the biosensor surface resulting from ligand induced changes in cell adhesion and spreading, toxicity, proliferation, and changes in protein-protein interactions near the plasma membrane. RWG optical biosensors have been used to detect changes in mass near the plasma membrane of cells following activation of G protein-coupled receptors (GPCRs), receptor tyrosine kinases, and other cell surface receptors. Ligand-induced changes in ion channel-protein interactions can also be studied using this assay. In this paper, we will describe the experimental procedure used to detect the modulation of Slack-B sodium-activated potassium (KNa) channels by GPCRs.

Optical biosensor techniques can detect changes in mass near the plasma membrane in living cells and allow one to follow cellular responses in both individual cells and populations of cells. This protocol will describe detection of the modulation of potassium channels by G-protein coupled receptors in intact cells using this approach.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

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., ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used method, but has the drawback that it requires fluorescent labeling, which can interfere with the activity of the molecules. Also, photobleaching and photodamage are concerns. In the case of microtubules, we have found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM). This suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro. In this paper, we show how a fluorescence microscope can be modified simply to obtain IRM images. IRM is easier and considerably cheaper to implement than other contrast techniques such as differential interference contrast microcopy or interferometric scattering microscopy. It is also less susceptible to surface defects and solution impurities than darkfield microscopy. Using IRM, together with the image analysis software described in this paper, the field of view and the frame rate is limited only by the camera; with a sCMOS camera and&#160;wide-field&#160;illumination&#160;microtubule length can be measured with precision up to 20 nm with a bandwidth of 10 Hz.&#160;

This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

There are several methods for visualizing purified biomolecules near surfaces. Total-internal reflection fluorescence (TIRF) microscopy is a commonly used ...