Name: stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope
Uploaded: 2018-07-03T14:00:00
Description: Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

We thank Margaret Gardel for generously allowing the use of the rheometer. We acknowledge support from the NIH (1R15GM116082) that enabled this work.

1. Fabrication of Soft Silicone Substrate
<ol>
	<li>Weigh out 1.75 g of the A component and 1.75 g of the B component (A:B = 1:1) from the soft silicone elastomer kit using (polystyrene) weighing trays.</li>
	<li>Add the A component to the B component in the weighing tray and mix them together for 5 min using an appropriate applicator stick.</li>
	<li>Add the above mixture to a 35 mm Petri dish. Allow the mixture to spread evenly across the Petri dish for a couple of minutes. 
	Note: The choice of the Petri dish d...

<ol>
	<li>Handorf, A. M., Zhou, Y., Halanski, M. A., Li, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+stiffness+dictates+development,+homeostasis,+and+disease+progression.">Tissue stiffness dictates development, homeostasis, and disease progression.</a> Organogenesis. 11 (1), 1-15 (2015).</li><li>Pelham, R. J., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+locomotion+and+focal+adhesions+are+regulated+by+substrate+flexibility.">Cell locomotion and focal adhesions are regulated by substrate flexibility.</a> Proceedings of the National Academy of Sciences. 94 (25), 13661-13665 (1997).</li><li>Lo, C. M., Wang, H. B., Dembo, M., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+movement+is+guided+by+the+rigidity+of+the+substrate.">Cell movement is guided by the rigidity of the substrate.</a> Biophysical Journal. 79, 144-152 (2000).</li><li>Discher, D. E., Janmey, P., Wang, Y. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+Substrate.">Tissue cells feel and respond to the stiffness of their Substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Kandow, C. E., Georges, P. C., Janmey, P. A., Beningo, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyacrylamide+hydrogels+for+cell+mechanics:+steps+toward+optimization+and+alternative+uses.">Polyacrylamide hydrogels for cell mechanics: steps toward optimization and alternative uses.</a> Methods in Cell Biology. 83, 29-46 (2007).</li><li>Johnston, I. D., McCluskey, D. K., Tan, C. K. L., Tracey, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+characterization+of+bulk+Sylgard+184+for+microfluidics+and+microengineering.">Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering.</a> Journal of Micromechanics and Microengineering. 24 (3), 035017 (2014).</li><li>Style, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+force+microscopy+in+physics+and+biology.">Traction force microscopy in physics and biology.</a> Soft Matter. 10 (23), 4047-4055 (2014).</li><li>Lee, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+cytoplasmic+domain+of+N-cadherin+reduces,+but+does+not+eliminate,+traction+force-transmission.">Deletion of the cytoplasmic domain of N-cadherin reduces, but does not eliminate, traction force-transmission.</a> Biochemical and Biophysical Research Communications. 478 (4), 1640-1646 (2016).</li><li>Frey, M. T., Engler, A., Discher, D. E., Lee, J., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+methods+for+measuring+the+elasticity+of+gel+substrates+for+cell+culture:+microspheres,+microindenters,+and+atomic+force+microscopy.">Microscopic methods for measuring the elasticity of gel substrates for cell culture: microspheres, microindenters, and atomic force microscopy.</a> Methods Cell Biol. 83, 47-65 (2007).</li><li>Lee, D., Rahman, M. M., Zhou, Y., Ryu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+confocal+microscopy+indentation+method+for+hydrogel+elasticity+measurement.">Three-dimensional confocal microscopy indentation method for hydrogel elasticity measurement.</a> Langmuir. 31 (35), 9684-9693 (2015).</li><li>Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+elastic+moduli+of+thin+layers+of+soft+material+using+the+atomic+force+microscope.">Determination of elastic moduli of thin layers of soft material using the atomic force microscope.</a> Biophysical Journal. 82 (5), 2798-2810 (2002).</li><li>Hertz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+die+Beru&#776;hrung+fester+elastischer+Ko&#776;rper.">&#220;ber die Beru&#776;hrung fester elastischer Ko&#776;rper.</a> Journal f&#252;r die reine und angewandte Mathematik. 92, 156-171 (1882).</li><li>Azioune, A., Carpi, N., Tseng, Q., Th&#233;ry, M., Piel, M., Cassimeris, L., Tran, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+micropatterns:+a+direct+printing+protocol+using+deep+UVs.">Protein micropatterns: a direct printing protocol using deep UVs.</a> Microtubules: In Vivo. , 133-146 (2010).</li><li>Bashirzadeh, Y., Qian, S., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-intrusive+measurement+of+wall+shear+stress+in+flow+channels.">Non-intrusive measurement of wall shear stress in flow channels.</a> Sensors and Actuators A: Physical. 271, 118-123 (2018).</li><li>Muhamed, I., Chowdhury, F., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+tools+to+study+cellular+mechanotransduction.">Biophysical tools to study cellular mechanotransduction.</a> Bioengineering (Basel). 4 (1), 12 (2017).</li><li>Dumbali, S. P., Mei, L., Qian, S., Maruthamuthu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+sheet-averaged+tension+within+a+large+epithelial+cell+colony.">Endogenous sheet-averaged tension within a large epithelial cell colony.</a> Journal of Biomechanical Engineering. 139 (10), 101008 (2017).</li></ol>

While the sphere indentation method is easy to implement, careful attention must be paid to the choice of indentor and the thickness of the soft silicone sample. The equation used to calculate the Young&#39;s modulus is valid under a set of conditions11and these are typically satisfied when the thickness of the silicone sample is &#62; 10% of the indentor radius and &#60; ~13x the indentor radius. We found that a silicone thickness of 5 - 10x the indentor radius was a good choice, wherein the samp...

Cells in soft tissues reside in a micro-environment whose stiffness is in the kilopascal range1, in contrast to tissue culture dishes whose stiffness is several orders of magnitude higher. Early experiments with cells on extracellular matrix protein-coated soft substrates showed that the substrate stiffness influences how cells move on as well as adhere to the extracellular matrix beneath2,3. In fact, the substrate stiffness fundamentally influences the cell function4&#160;in a manner similar to pervasive biochemical signals. Polyacrylamide gels (coated with extracellular matrix proteins) are (water-permeating) hydrogels that have been extensively used as cell culture substrates for mechanobiology studies5. Polydimethylsiloxane (PDMS), the most common silicone (polysiloxane), has been widely used as a stiff silicone with megapascal-range stiffness for micron-scale fabrication6. More recently, soft silicone substrates with stiffness in the more physiologically relevant kilopascal range have been employed as cell culture substrates for mechanobiology studies7,8.
Several methods have been used to measure the stiffness of flexible substrates, including atomic force microscopy, macroscopic deformation of whole samples upon stretching, rheology, and indentation using spheres and spherically tipped microindentors9. While each technique has its own advantages and disadvantages, indentation with a sphere is an especially simple yet fairly accurate method that only requires the access to a widefield fluorescence microscope. Indentation with a metallic sphere has been used to measure the stiffness of hydrogels in prior work3,9,10. Early work that demonstrated the importance of substrate stiffness to cell movement utilized this method to determine hydrogel substrate stiffness3. More recently, confocal microscopy has also been used for an elegant characterization10.
Here, we present a step-by-step protocol for preparing a soft silicone substrate, coupling fluorescent beads (and an extracellular matrix protein such as collagen I) just to the top surface, imaging an indenting sphere and the top surface using phase and fluorescence imaging, respectively, and finally analyzing the images to compute the Young&#39;s modulus of the silicone substrate. The soft silicone substrate prepared in this manner can be readily used for traction force microscopy experiments. The use of stiff silicone (instead of a Petri dish) as the base for the soft silicone also enables mechanobiology studies using an external stretch. Where warranted, practical considerations necessary for avoiding possible complications are also indicated.

<table border="0" cellpadding="0" cellspacing="0" width="949">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:353px;">CY 52-276 A/B silicone elastomer kit</td>
			<td style="width:191px;">&#160;Dow Corning</td>
			<td style="width:144px;">CY 52-276</td>
			<td style="width:263px;">Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific Pierce EDC</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td>PI22980</td>
			<td>Store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#160;Pierce Sulfo-NHS crosslinker</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td>PI-24510</td>
			<td>Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carboxyl fluorescent pink particles, 0.4-0.6 &#181;m, 2 mL</td>
			<td>Spherotech, Inc.</td>
			<td>CFP-0558-2</td>
			<td>Store at 4&#176;C, do not freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.0 mm Acid washed Zirconium beads</td>
			<td style="width:191px;">OPS Diagnostics LLC</td>
			<td>BAWZ 1000-250-33</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:353px;">Deep UV chamber with ozone evacuator</td>
			<td style="width:191px;">Novascan Technologies, Inc.</td>
			<td style="width:144px;">PSD-UV4, OES-1000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Wide field fluorescence microscope</td>
			<td style="width:191px;">Leica Microsystems</td>
			<td style="width:144px;">DMi8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Collagen I, from rat tail</td>
			<td style="width:191px;">Corning</td>
			<td style="width:144px;">354236</td>
			<td>Stock concentration = 4 mg/ml; store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">ImageJ-NIH</td>
			<td style="width:191px;">N/A</td>
			<td style="width:144px;">N/A</td>
			<td>public-domain software</td>
		</tr>
	</tbody>
</table>

stiffness measurement of soft silicone substrates for mechanobiology studies using a widefield fluorescence microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Using the protocol detailed above, we prepared soft silicone in a 35 mm Petri dish, cured it at 70 &#176;C for 30 min and coupled fluorescent microbeads (and collagen I) to the top surface as schematically depicted in Figure 1. Deep UV has been used previously for the eventual protein coupling to substrates13. Note that (I) the curing conditions used here are specific to this soft silicone and (II) the indentation measurement is pe...

Watch this Scientific Journal Video about Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope at JoVE.com

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

This method can be used to categorize substrates used to study key phenomena in mechanobiology, the cell migration and first generation. The main advantage of this technique is that it requires the use of a wide-field microscope, a piece of equipment that is commonly available in many labs. This method can be applied to measure the Young's modulus of silicon or polyacrylamide gel, as well as any of the soft isotropic linear elastic material.The substrate's prepared and characterized by this method can also be used for self-stretching experiments with the stiff silicone base. To fabricate the soft silicone substrate after weighting out about 1.75 grams each of components A and B from the soft silicone elastomer kit, add most of the weighed portion of component A to the B component polystyrene weighing tray and use an appropriate applicator stick to mix the two components together for five minutes. Transfer the mixture to a 35 millimeter Petri dish and allow the substrate to spread evenly across the Petri dish for a few minutes.Next, place the Petri dish in a vacuum chamber for 15 minutes to remove any air bubbles and pre-heat a hot plate. When the hot plate reaches 70 degrees Celsius, place a glass slide onto the hot plate, and place the Petri dish onto the glass side. Then, let the silicone cure at 70 degrees Celsius for 30 minutes.To couple the soft silicone with fluorescent beads, place the cured silicone in a deep UV chamber, five to ten centimeters away from the UV lamp and expose the sample to deep UV light for five minutes. While the silicone is being treated, dissolve 19 milligrams of EDC in 500 microliters of deionized water in a 1.5 milliliter microcentrifuge tube. And vortex the tube to dissolve the chemical.Next, add 500 microliters of deionized water to 11 milligrams of sulfo-NHS in a second 1.5 milliliter tube. And vortex to dissolve the chemical. Then, combine the EDC and sulfo-NHS solutions into a single microcentrifuge tube.Now, add 30 microliters of an appropriate suspension of fluoro-4 conjugated carboxylate-modified micro-beads and 0.02 milligrams of rat tail Collagen 1 to the mixture. After briefly vortexing, place a piece of Parafilm onto the lid of a second smaller diameter lid and transfer one milliliter of the EDC-NHS bead Collagen 1 mixture to the Parafilm. Invert the Petri dish containing the soft silicone over the mixture, supporting the dish on both sides with two glass slides, so that the soft silicone surface contacts the bead solution without directly touching the surface of the smaller diameter lid below.Then, cover the sample with aluminum foil for 30 minutes at room temperature, before washing the silicone two times, with two milliliters of PBS per wash and let the silicon cure upright in two milliliters of fresh PBS at 37 degrees Celsius overnight. The next morning, use pointed tweezers to drap five millimeter zirconium sphere indentors onto the soft silicone under at least one milliliter of PBS away from the edges of the silicone layer and at least five indentor diameters away from the location of the other indentors. Next, place the dish of soft silicon onto a wide-field microscope stage and use the 10x objective to obtain a phase image of a whole indentor without any visible defects.After saving the image, select the appropriate fluorescent channel for visualizing the fluorescent micro-beads. With the indentor center near the right edge of the frame, decrease the z-coordinates until the fluorescent micro-beads under the sphere indentor's center go just out of focus. Then, acquire a z-stack with an image for every 0.5 micrometer z-increment until the micro-beads in the top layer of the silicone near the left edge of the imaging frame come in and then out of focus.To calculate the silicone stiffness, first, open the indentor phase image in ImageJ and click analyze and set scale and check unit of length to confirm that the length is set to pixels. Using the line tool, click and hold onto a point on the indentor edge, moving the cursor to a point on the diametrically opposite edge, to measure the diameter of the indentor and note the length in pixels on the status bar of the ImageJ main window before releasing the cursor. Next, open the file menu and select import an image sequence to allow an image in the z-stack of fluorescent micro-bead images to be selected and click okay to open the stack.Using the line tool, draw a line across a well-defined micro-bead in one image and click analyze plot profile in live to obtain the updated line scan intensity across the bead, while selecting different frames. The frame that gives the highest value of the maximum intensity can then be selected as the frame in focus. Fluorescence images of the micro-beads in the top surface of the silicone taken at an x-y frame position, places the region under the indentor to the far-right side of the frame.The region on the far-left side of the frame is then selected as the region away from the indentor. In this representative set of intensity line scans measured across a single micro-bead, the focus varies in z-increments of 0.5 micrometers. The z-value corresponding to the in-focus image can then be objectively selected based on the z-value corresponding to the line scan with the highest maximum intensity.Here, the Young's modulus of the silicone for various A:B component mixture ratios, was obtained based on the method as just demonstrated. After watching this video, you should have a good understanding of how to prepare soft silicone substrates, cup the fluorescent beads to them, and measure the stiffness of the substrate using a wide-field fluorescent microscope.

stiffness-measurement-soft-silicone-substrates-for-mechanobiology

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

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 ...

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 ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

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

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...