Name: pattern generation for micropattern traction microscopy
Uploaded: 2022-02-17T14:00:00
Description: Watch this Scientific Journal Video about Pattern Generation for Micropattern Traction Microscopy at JoVE.com

The authors would like to thank Dr. Paul Barbone from the Boston University Department of Mechanical Engineering for helpful discussions and assistance with data analysis. This study was supported by NSF grant CMMI-1910401.

1. Creation of silicone masters
NOTE: Most of the process of the design, creation, and troubleshooting of silicon masters for the repeated molding of PDMS stamps has been covered previously21, so only key differences in this new approach will be described here.
<ol>
	<li>Create the design for the photomask using AutoCAD or similar design software. Coat one side of the photomask, a thin piece of glass, with a thin layer of chrome to control UV light sc...

An improved method of indirectly patterning PAA hydrogels is described in this paper. This approach builds on methods that have been used previousely20,35,36,37,38,39,40,41,42. The primary change is that PDMS stamps are no...

Most cell phenotypes exert traction forces on their environment. These traction forces are generated by a cell&#39;s contractile cytoskeleton, which is a network of actin and myosin, and other filamentous biopolymers and crosslinking proteins1,2,3,4. Forces generated within the cell can be transmitted to the extracellular environment or adjacent cells, primarily via transmembrane proteins such as integrins and cadherins, respectively5,6. How a cell spreads or contracts-and the magnitudes of the traction forces associated with those movements-is the result of an intimate conversation with its environment, which largely depends on the type and quantity of protein present in the extracellular matrix (ECM)7,8 and the stiffness of the ECM. Indeed, traction force microscopy has become an invaluable tool for understanding cell responsiveness to local stimuli such as substrate stiffness, imposed mechanical stresses and strains, or contact with other cells. This information is directly relevant to the understanding of diseases such as cancer and asthma9,10,11,12.
A system that can be used to measure force-induced deformation of a substrate of known material properties is required to calculate traction forces. These changes must be tracked over time, requiring both imaging and image processing techniques. One of the first methods used to determine cellular traction forces was the observation and analysis of the contraction of collagen hydrogels seeded with cells, though this method was only semiquantitative13. Another, more refined method was to measure the traction forces exerted by single cells by determining the forces resulting from the deformation of a thin sheet of silicone14. Later on, more quantitative measurement techniques were developed, and these methods also allowed for the use of soft hydrogels such as polyacrylamide (PAA)12,15,16. When using these soft materials, traction forces could be determined from the force-induced displacement of randomly displaced beads embedded in the hydrogel and the mechanical properties of the gel16,17. Another advancement came with the development of micropost arrays made of soft polydimethylsiloxane (PDMS) so that their deflection could be measured and converted to force using the beam theory18.
Finally, methods for micropatterning soft hydrogels were developed as these approaches allow control of the contact areas for cell adhesion. By measuring the deformation of the micropattern within a cell&#39;s contact area, traction forces could easily be calculated because a force-free reference image is not required19. This method has been widely adopted as it allows for the indirect patterning of a regular array of micron-sized, discrete fluorescent protein adhesion points onto PAA gels for the measurement of cellular traction forces20. To calculate these forces, an image-processing algorithm, which can track the movements of each micropatterned dot without requiring user input, has been developed21.
While this method is simple for creating entire grids of dot patterns, it is more complicated when patterns of isolated patches (or islands) of dots are desired. Micropatterned islands are useful when control of shape, and to some extent of size, of clusters of cells is needed. To create these islands, the aforementioned method of microcontact printing necessitates two distinct steps: i) using one PDMS stamp to create a high-fidelity pattern of dots on a coverslip, and then ii) using a second different PDMS stamp to remove most of those dots, leaving behind isolated islands of dots21. The difficulty in creating islands with this original method is compounded by the fact that making consistent grid patterns in the first step of the process is challenging on its own. Microprinting stamps are composed of an array of circular microposts, the diameter of which corresponds to the desired dot size. These stamps are then coated with an even layer of protein and then stamped with a precise amount of pressure onto treated coverslips to create the desired pattern. On the one hand, applying too much pressure to the stamp can result in uneven protein transfer and poor pattern fidelity due to pillar buckling or sagging between pillars, leading to contact with the glass. On the other hand, applying too little pressure results in little to no protein transfer and poor pattern fidelity. For these reasons, a transfer process that can be used to consistently create high-quality micropatterns of isolated islands of dots in just one step is desired.
Herein, a method is described for the indirect micropatterning of islands of micron-sized fluorescent protein adhesion points onto a PAA gel that is more consistent and versatile than previously developed methods. Whereas older indirect micropatterning methods rely on the transfer of protein patterns from a PDMS stamp to an intermediate substrate, the method introduced here uses PDMS stamps instead as a vessel for protein removal, not addition. This is done by first fundamentally changing the structure of the PDMS stamps used. Rather than making stamps that are composed of a pattern of evenly spaced circular pillars, stamps are made up of a pattern of evenly spaced circular holes in this method.
With this new structure, the surface of these PDMS stamps can then be treated with glutaraldehyde as described previously20,29,30, making the stamp able to bond covalently with protein. When used on a glass coverslip evenly coated with fluorescent protein, these glutaraldehyde-treated PDMS stamps are used to remove most of the protein on the surface of the coverslip, leaving behind only the desired pattern of dots predetermined by the location of micron-sized holes on the stamp. This change increases the success rate for generating patterns made up of a near-continuous grid of dots and for creating isolated islands of dots through only one step.

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			<td>(3-aminopropyl)trimethoxysilane</td>
			<td>Sigma Aldrich</td>
			<td>#281778</td>
			<td></td>
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			<td>1.5 mL Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>#05-408-129</td>
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			<td>15 mL conical tube</td>
			<td>Fisher Scientific</td>
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			<td>4 x 4 in 0.060 Quartz LR Chrome Photomask</td>
			<td>Advance Reproductions Corporation</td>
			<td>N/A</td>
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			<td>Acrylamide Solution, 40%</td>
			<td>Sigma Aldrich</td>
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			<td>AlexaFluor 488</td>
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			<td>#A20000</td>
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			<td>Aminonium Persulfate</td>
			<td>Fisher Scientific</td>
			<td>#BP179-25</td>
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			<td>Bisacrylamide</td>
			<td>Fisher Scientific</td>
			<td>#PR-V3141</td>
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			<td>Ethanol</td>
			<td>Greenfield Global</td>
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			<td>Glass Coverslips, 25 mm round</td>
			<td>Fisher Scientifc</td>
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			<td>Glass Coverslips, 30 mm round</td>
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			<td>Hamamatsu ORCA-R2 Camera</td>
			<td>Hamamatsu</td>
			<td>#C10600-10B</td>
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			<td>Human Plasma</td>
			<td>Valley Biomedical</td>
			<td>#HP1051P</td>
			<td>Used to isolate fibronectin</td>
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			<td>Hydrochloric Acid, 1.0 N</td>
			<td>Millipore Sigma</td>
			<td>#1.09057</td>
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			<td>ImageJ</td>
			<td>Wayne Rasband</td>
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			<td>Interchangeable Coverslip Dish Set</td>
			<td>Bioptechs</td>
			<td>#190310-35</td>
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			<td>Kim Wipes</td>
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			<td>#06-666-11C</td>
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			<td>Mask Alinger</td>
			<td>Karl Suss</td>
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			<td>Mathworks</td>
			<td>#R2021a</td>
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			<td>MetaMorph Basic</td>
			<td>Molecular Devices</td>
			<td>#v7.7.1.0</td>
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			<td>N-hydroxysuccinimide ester</td>
			<td>Sigma Aldrich</td>
			<td>#130672-5G</td>
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			<td>NucBlue Live Cell Stain</td>
			<td>Thermo Fisher</td>
			<td>#R37605</td>
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			<td>Olympus IX2-ZDC Inverted Microscope</td>
			<td>Olympus</td>
			<td>#IX81</td>
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			<td>PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>#52-1308-00</td>
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			<td>Photoresist Spinner Hood</td>
			<td>Headway Research</td>
			<td>#PWM32</td>
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			<td>Plasma Cleaner</td>
			<td>Harrick</td>
			<td>#PDC-001</td>
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			<td>Plasma Etcher</td>
			<td>TePla</td>
			<td>#M4L</td>
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			<td>Prior Lumen 200Pro Light Source</td>
			<td>Prior Scientific</td>
			<td>#L200</td>
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			<td>Silicon Wafers, 100 mm</td>
			<td>University Wafer</td>
			<td>#809</td>
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			<td>SU-8 2005</td>
			<td>Kayaku Advanced Materials Inc.</td>
			<td>#NC9463827</td>
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			<td>SU-8 Developer</td>
			<td>Kayaku Advanced Materials Inc.</td>
			<td>#NC9901158</td>
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			<td>Sylgard 184 Silicone Elastomer</td>
			<td>Essex Brownell</td>
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			<td>Tetramethylethylenediamine</td>
			<td>Fisher Scientific</td>
			<td>#BP150-20</td>
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			<td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td>
			<td>Sigma Aldrich</td>
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			<td>UAPON-40XW340 Objective</td>
			<td>Olympus</td>
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			<td>UV Flood Exposure</td>
			<td>Newport</td>
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			<td>Wafer Carrier Tray, 110 x 11 mm</td>
			<td>Ted Pella, Inc.</td>
			<td>#19395-40</td>
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pattern generation for micropattern traction microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

PAA hydrogels with the Young&#39;s modulus of E = 3.6 kPa and the Poisson&#39;s ratio of &#957; = 0.445 were made for use by this subtractive micropatterning method. The hydrogels were made to be ~100 &#181;m thick, which allows them to be imaged with the imaging setup used here while also preventing the cells from sensing the rigid coverslip below the gel, which would cause problems in studies focused on cellular rigidity sensing23,33. Gels of ...

Watch this Scientific Journal Video about Pattern Generation for Micropattern Traction Microscopy at JoVE.com

Pattern Generation for Micropattern Traction Microscopy

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

This protocol allows for the consistent creation of high-fidelity protein micro patterns of whatever shape is desired, notably isolated islands of pattern for study of cell clusters. This technique can be used to make isolated island micro patterns of any shape and size in only one step, whereas previously making such patterns required two separate steps. Begin by mixing PDMS in the correct curing agent to base ratio as described in the manufacturer's instructions.Incubate at room temperature and pressure for 15 minutes, then degas the mixture under vacuum for 15 minutes. Pour the PDMS into the master mold and transfer it to an incubator set to 37 degrees Celsius to cure overnight. Remove the master mold from the incubator and allow it to cool down to room temperature.While the master mold is cooling down, sonicate 25 millimeter coverslips in ethanol for 10 minutes. Use as many coverslips as the number of stamps being prepared. Thoroughly rinse the coverslips with DI water and dry them using a filtered air gun.Treat the coverslips with plasma for one minute using the plasma cleaner under a high vacuum. Slowly release the vacuum after treatment to prevent the coverslips from moving inside the chamber. Coat each coverslip with 100 microliters of the fluorescent labeled protein solution in a room devoid of direct sunlight or overhead lighting.Cover the samples for extra protection from light and incubate them for 20 minutes at room temperature. Thoroughly rinse each coverslip with DI water. Remove excess water from the surface by gently tapping the sides of each coverslip onto a paper towel or a similar absorbent material.Allow the coverslips to dry completely by leaving them uncovered in the dark for at least 30 minutes. While the protein coated coverslips dry, remove the PDMS stamp from the master mold by cutting it with a scalpel or any sharp blade. Treat the PDMS stamps with plasma for two minutes under a high vacuum.Place the stamps in a container with a lid under the fume hood, then coat each stamp with a thin layer of 10%3-APTMS diluted in 100%ethanol. Cover the container with the stamps and incubate at room temperature for five minutes. Thoroughly rinse each stamp on both sides with DI water.Place the stamps in a clean container and coat them with 2.5%glutaraldehyde prepared in DI water. Cover the stamps and incubate them at room temperature for 30 minutes. Rinse the stamps thoroughly with DI water.Remove excess water from the surface as previously described and allow the stamps to dry uncovered for 30 minutes. If after 30 minutes the protein-coated coverslips or the stamps are not completely dry, use a filtered air gun to dry them completely. Push the pattern side of the stamps onto the coverslips with enough pressure for them to make complete contact.Leave it undisturbed for 15 minutes. Then carefully peel the PDMS stamps off the coverslips. Using a fluorescent microscope with the appropriate filter, check the fidelity of the patterned coverslips.Use the patterned coverslips immediately or store them away from direct light until further use. Before preparing the PAA hydrogel precursor, remove the acrylic acid NHS ester from the refrigerator to reach room temperature before being opened. Prepare an interchangeable coverslip dish set by sterilizing it with 70%ethanol, then incubate it under UV light in a biosafety cabinet for at least 30 minutes before use.Under the fume hood, add 1.25 milliliters of 40%acrylamide prepared in DI water to a 15 milliliter conical tube. To the same tube, add 175 microliters of bis-acrylamide solution prepared in DI water. Then add 500 microliters of 10X PBS, followed by 2.915 milliliters of DI water.Pipette 969 microliters of this precursor into a 1.5 milliliter microcentrifuge tube and store the rest at four degrees Celsius for up to two weeks. Measure 50 to 100 milligrams of APS in a fresh microcentrifuge tube and dilute it to 100 milligrams per milliliter in DI water. Open the NHS ester in the hood and carefully measure up to three milligrams of NHS in a microcentrifuge tube.Dilute the NHS ester to one milligram per milliliter in 1X PBS. Under the fume hood, add two microliters of TEMED to the microcentrifuge tube containing the 969 microliter aliquot of PAA precursor. Add 15 microliters of one molar HCL to decrease the pH of the hydrogel solution and avoid hydrolysis of the NHS ester.Add 10 microliters of the NHS ester solution to the tube. Perform the following steps in a biosafety cabinet. Carefully place the 30 millimeter coverslip within the middle part of the coverslip dish set with the 3-APTMS and glutaraldehyde treated side up and screw the plastic ring on top.Set up the patterned coverslip with minimum light exposure to prepare for the next step. Pipette five microliters of APS solution into the PAA precursor aliquot tube, then invert and mix. Immediately pipette 35 microliters of this solution onto the 30 millimeter coverslip.Place the patterned coverslip on the solution with the protein side down. Be careful not to create air bubbles in the hydrogel. Protect the hydrogel from light and allow it to polymerize for 90 minutes.Once the hydrogel has polymerized, use a razor blade or scalpel to remove the upper coverslip, ensure that the coverslip does not fall back or slide off the gel once removed to avoid ruining the pattern on the surface of the gel. To passivate any remaining NHS ester in the hydrogel, add two milliliters of sterile PBS to the gel and incubate at 37 degrees Celsius for 45 minutes. Immediately prepare the gels for experiments or store them overnight in sterile PBS at four degrees Celsius until further use.The hydrogels were indirectly patterned with fluorescent fibronectin to visualize and measure cellular traction forces within clusters and create island patterns of predetermined size and shape. The pattern quality was directly related to the fidelity of the master mold from which the PDMS stamp was cast. This method could fabricate isolated, well-defined island patterns of predetermined shape.The fibronectin adhesion dots were present only within the desired area of the island. These isolated islands of adhesion dots further allowed better control of cluster shape. Coating PDMS stamps with two little 3-APTMS is critical because it will limit the effectiveness of the removal method, while too much will create an orange residue on the stamp surface.The patterns created here can be used to determine cellular traction forces in both individual cells and clusters, which gives insight into their ability to maintain mechanical homeostasis.

pattern-generation-for-micropattern-traction-microscopy

Research

JoVE Journal

Bioengineering

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Pattern-based Search of Epigenomic Data Using GeNemo

Compared with the robust text-based search tools for genomic or RNA sequencing data, current methodologies for pattern-based searches of epigenomic and other functional genomic data are very limited. GeNemo is the first online search tool that accomplishes this goal. Users input their functional genomic data in the Browser Extensible Data (BED), Peaks, and bigWig formats, and may search for data in any of the three formats. Users may specify which types of datasets to search against, choosing from a variety of online datasets, with the Encyclopedia of DNA Elements (ENCODE) representing different epigenomic marks, transcriptional factor binding sites, and chromatin hypersensitivities or accessibilities in specific cell types, and developmental stages or species (mouse or human). GeNemo returns a list of genomic regions with matching patterns to the input data, which may be viewed in the browser as well as downloaded in the BED file format. The upgraded GeNemo has improved graphical display, has more robust interface, and is no longer prone to errors due to changes in the University of California, Santa Cruz (UCSC) genome browser. Troubleshooting steps for common problems are discussed. As the amount of functional genomic data is expanding exponentially, there is a critical need to develop and refine new bioinformatic tools such as GeNemo for data analyses and interpretation.

Unlike DNA sequence data, epigenomic data are not readily subjected to text-based searches. Presented here are the procedures to use an upgraded version of GeNemo, a web-based bioinformatics tool, to conduct pattern-based searches for similarities in epigenomic data comparing available online databases including Encyclopedia of DNA Elements with user&#39;s data.

Compared with the robust text-based search tools for genomic or RNA sequencing data, current methodologies for pattern-based searches of epigenomic and ...

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 ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores&#8217; germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that &#62;80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores&#8217; 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores

Germinant receptor proteins cluster in &#8216;germinosomes&#8217; in the inner membrane of Bacillus subtilis spores. We describe a protocol using super resolution microscopy and fluorescent reporter proteins to visualize germinosomes. The protocol also identifies spore inner membrane domains that are preferentially stained with the membrane dye FM4-64.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence ...