Name: easy and accurate mechano-profiling on micropost arrays
Uploaded: 2015-11-17T15:00:00.000+00:00
Duration: 10 min 25 s
Description: Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

This work was supported by the Committee Technology and Innovation (CTI) Switzerland grant 14796-PFLS-LSCTI. MicroPost arrays were generously provided by MicroDuits GmbH.

1. Culturing Cells on Micropost Arrays
Note: All steps must be performed in a biosafety cabinet to ensure sterility. The volumes given here are for T25 cell culture flasks. The cell culture medium recipe and cell seeding density are optimized for bone cancer cell lines HuO9 and M132.
<ol>
	<li>Prepare a cell culture for seeding onto a micropost array.
	<ol>
		<li>Inspect the cell culture quality by placing the culture flask on a standard light microscope. Ensure that the cells show a typical growth and morphology by estimating how much of the culture flask bottom is covered. A coverage of 70%-80% reflects a healthy growth rate. Pay attention to the amount of floating cells as they represent dead cells and/or an overgrown culture.</li>
		<li>Trypsinize cells by removing medium and washing with 4 ml 1x Phosphate buffered saline (PBS) buffer. Add 0.8 ml 1x Trypsin/Ethylenediaminetetraacetic acid (EDTA) and incubate at RT until the cells dislodge, which takes about 2-4 min. Check the process with the microscope and occasionally tap the flask gently to support the dislodging.</li>
		<li>Add 5 ml cell culture medium (Dulbecco&#8217;s modified Eagle medium (DMEM)/F12 with 10% fetal bovine serum (FBS), 1% penicillin/streptavidin (Pen/Strep)) to stop the reaction and to wash off residual cells that still sit on the flask&#8217;s bottom. Subsequently pipette this suspension up and down several times to gain a homogenous mixture. Make sure to pipette all solutions across the original growth area of the flask to gain an effective dislodging.</li>
		<li>Transfer the cell suspension in to a 15 ml tube and centrifuge it using a table top centrifuge at 0.5 x g for 3 min.</li>
		<li>Remove the supernatant and re-suspend the cell pellet in 5 ml medium by pipetting up and down 5 times. Make sure to avoid the generation of bubbles while doing so.</li>
		<li>Determine cell density by using a cell counting chamber and a light microscope. Check the cell suspension in cell counting chamber for cell aggregates and ensure to have only single cells for the subsequent experiment. Pipette again up and down several times if the cells tend to form aggregates to separate them.</li>
		<li>Dilute the cell suspension with sufficient medium to gain a cell solution of 25,000 cells/ml. Mix it well by inverting the closed tube several times.&#160;</li>
	</ol>
	</li>
	<li>Prepare a micropost array for cell seeding. 
	Critical Note: Do not pipette onto the micropost array directly as this could potentially harm the microposts, especially when unwanted bubbles are introduced. Furthermore, make sure that the micropost array never dries out as occurring capillary forces lead to collapsing of the microposts.
	<ol>
		<li>Place the glass substrate with the micropost array face up in a well of a 12-well plate by using a pair of tweezers and wet it by adding 1 ml ethanol (99%). Incubate at RT for 20-30 sec.</li>
		<li>Dilute the ethanol stepwise by adding approximately 1 ml sterile de-ionized water (DI water) on the well side and aspirating roughly 1 ml using a transfer pipette or aspiration device. Repeat this step at least 3 times.</li>
		<li>Replace the DI-water with PBS-buffer in the same manner by adding and aspirating approximately 1 ml. Repeat this step 3 times.</li>
		<li>Replace the PBS-buffer with medium by adding and aspirating approximately 1 ml of medium. Repeat this step 3 times.</li>
	</ol>
	</li>
	<li>Pipette 1 ml cell solution (25,000 cells) on top of each prepared micropost array. Close multi-well plate and transfer it to an incubator (CO2 5%, 37 &#176;C). Let the cells adhere and grow on top of microposts for 6&#160;-7 hr.</li>
	<li>Inspect the adhesion process occasionally by using a light a microscope. Check that most of the cells appear spread out across multiple microposts.</li>
</ol>
2. Fixing &#38; Staining Cells
Note: All steps and volumes given here are for a single well of a 12-well plate. It is recommended to process not more than four of the twelve wells at a time to avoid a drying out of the wells after aspiration of any liquid, which will result in a collapse of the microposts.
<ol>
	<li>Aspirate medium from the well and wash cells 2x with 1 ml PBS-buffer. Make sure to apply a gentle force while washing with PBS to remove cell debris that accumulated on the micropost array during incubation and to detach dead cells.</li>
	<li>Fix the cells with 0.5 ml 3.7% buffered formaldehyde solution for 5 min.</li>
	<li>Replace the formaldehyde solution by washing the micropost array 2x with 1 ml sterile DI water.</li>
	<li>Stain the cells with dye (0.05% Coomassie Brilliant Blue in 50% water, 40% ethanol and 10% acetic acid) for about 90 sec. Wash excess staining solution off 2x with 1&#160;ml sterile DI water. Add 1 ml DI water to micropost array.</li>
	<li>Check staining result using a light microscope. Repeat the staining step, if cell body is too faint to be visualized.</li>
	<li>Store micropost arrays with the stained cells on top in a refrigerator at 4 &#176;C. Ensure to keep them under water at all times.</li>
</ol>
3. Cell Imaging
Note: Step 4 only applies to microscopes without infinity corrected optics.
<ol>
	<li>Add 2 ml DI-water to a Petri dish with a thin glass bottom for high resolution imaging.</li>
	<li>Transfer micropost array using a pair of tweezers into the imaging dish with the microposts facing up.</li>
	<li>Place the imaging Petri dish on a moveable stage of a light microscope.</li>
	<li>Turn the compensation ring of the lens used for imaging until the number on the scale represents the total thickness of all materials along the optical path to compensate for the mismatch in refractive indices of the glass substrate, the silicone elastomer and the liquid on top. This should lead to a bright appearance of the microposts cores.</li>
	<li>Close the iris on the illumination side down to 50% and remove any phase contrast rings from the optical path enabling an ordinary bright-field mode.</li>
	<li>Align the micropost array that the microposts form a horizontal line across the observation field. Define a start point (e.g., top left) and move the Petri dish stepwise across the stage scanning the micropost array while taking numerous images.</li>
	<li>Take all images with the highest resolution setting for the camera using a 20X or 40X objective. Aim to have a single cell in the center of the image.</li>
	<li>Sweep along the z-axis through the substrate until the micropost tips are in focus. Use the fine tuning focus wheel of the microscope and turn it towards focusing on the micropost bottom for about 2-3 &#181;m.</li>
	<li>Establish a standard imaging procedure by taking series of test images of one array section sweeping through the imaging parameters one at a time (e.g., exposure time, iris setting, lamp settings etc.). Analyze images with MechProfiler and determine a suitable parameter set for a fast and reliable analysis.</li>
</ol>
4. Image Analysis with Open Source Software &#8220;MechProfiler&#8221;
Note: All software functions can be activated with a pointing device using the left mouse button. However, a trained operator will use the documented shortcut keys designed to be on the left half of the keyboard to enable an efficient two-handed image processing.
<ol>
	<li>Start image analysis software MechProfiler and open a range of images that need to be analyzed by clicking &#8220;Open&#8221;.</li>
	<li>Insert all parameters into the &#8220;Settings&#8221; section given by the software developer. Determine the parameters that must be custom configured (i.e., contour threshold and minimal distance) according to the procedures described in detail by the software manual.</li>
	<li>Analyze images one-by-one by cropping them to the area of interest by using &#8220;Crop&#8221; (click and drag with the mouse button pressed down). Double click inside the drawn rectangle to finish this action.</li>
	<li>Marking each visible cell outline one-by-one by clicking on &#8220;Draw cell outlines&#8221; and use the cross hair cursor for drawing including all microposts the cell is attached to.</li>
	<li>Discard any unwanted microposts by clicking &#8220;Discard Posts&#8221; and use the cross hair cursor for drawing. Enclose all microposts that belong to a cell outside of the image section or that are deflected for any other reason but not by the cell of interest.</li>
	<li>Start the software subroutine &#8220;Find Centroids&#8221; by mouse click. Adjust the filter setting right next to the &#8220;Find Centroids&#8221; button until all microposts are registered, which is visible by a red cross in their center. If the filter setting is too low microposts will be ignored, if it is too high multiple positions will be marked for a single micropost. Keep the filter setting constant during an analysis session.</li>
	<li>Use the manual editing function &#8220;Manual Edit&#8221; for centroids with multiple or missed micropost positions. Use the mouse cross hair to select the micropost in question by clicking and dragging it across. Double click inside the rectangle and place the missing red marker in the enlarged image section, which automatically closes. Discard such a micropost if it is outside the drawn cell outline (see step 4.5) before proceeding.</li>
	<li>Find the ideal micropost grid by activating the &#8220;Generate Grid&#8221; function with a mouse click. Ensure the position corresponds with the true micropost head, shown by a blue ring, inside the drawn cell outline.</li>
	<li>Correct any misplaced grid maker inside the cell area where needed using the corresponding &#8220;Manuel Edit&#8221; function with a mouse click. Use the cross hair to select the micropost in question by clicking and dragging it across. Double click inside the appearing rectangle and place the missing blue marker in the enlarged image section, which automatically closes.</li>
	<li>Use the button &#8220;Calculate Deflections&#8221; by mouse click to get a histogram of the calculated deflection values based on the difference between a micropost&#8217;s position inside the image section and the generated ideal grid.</li>
	<li>Save the complete analysis including the tables of values by a mouse click on &#8220;Save&#8221;.</li>
	<li>Continue the image analysis either by clicking on &#8220;Reset View&#8221; and analyze another image section or mouse click on &#8220;Next Image&#8221;, which conveniently can be done using the right keyboard curser key.</li>
</ol>
5. Data Analysis &#8212; Mechanoprofiling
<ol>
	<li>Open the file &#8220;results.xls&#8221; with an office spreadsheet program from the folder with the analyzed images. It contains the data with all calculated values including all micropost deflections and standard derivative measures such as work (i.e., substrate deformation energy) by the analyzed cell.</li>
</ol>

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The lead author (NG) is co-founder of MicroDuits GmbH, the commercial supplier of the micropost arrays used in this study. No other authors have any potential conflicts of interest with this work.

This work seeks to advance the field of traction force microscopy by substantially lowering technical and practical barriers to entry. These barriers come from two sides. First and foremost are the numerous non-trivial technical challenges that must be overcome to reproducibly manufacture and experimentally commission a micropost array. Second, is the similarly non-trivial need for a reliable semi-automated analysis of single cell forces &#8211; keeping in mind that a typical experiment may require the analysis of hundreds or even thousands of cells. The above described techniques were developed to make micropost based traction force microscopy accessible within cell biology laboratories that do not have resident engineering expertise. The sensors and analysis techniques presented in this article should allow non-experts to readily and accurately analyze the forces exerted by single cells.

Apart from access to a cell biology lab with at least a mid-grade bright-field microscope, the described methods require few additional technical elements. Here most users can perform valid traction force microscopy experiments with even limited experience, despite the power and versatility of the technique. Nonetheless there are three critical aspects in the presented protocol that must be considered. First it is necessary to ensure that the micropost arrays are always covered with liquid to avoid that their collapse due to capillary forces. Second one needs to optimize imaging parameters for acquired micropost images, which is best done by taking series of images of the same array position while varying exposure time, iris aperture, and compensation ring position. It is important to find the correct focal plane for the micropost tip, and this aspect does take some experience given the small dimensional depth of the sensor arrays. Third one must download the openly accessible image analysis software MechProfiler discussed here, for which a detailed user manual and training examples are available at the download site. After some experience using the software to analyze examples provided with the manual, the user should take test images on the imaging setup to be used so as to configure suitable microscope settings. Based on the obtained images, the contour threshold value should be similarly optimized. A single micropost image can be analyzed by a trained user within a minute using the open-source MechProfiler software regardless of whether it contains one or more cells, allowing the generation of statistically relevant results (analysis of several hundreds of cells) within a few hours.
Another aspect that requires some tuning is the optimization of any applied cell staining procedure. When cells are overly-stained the software can fail to register the correct micropost positions. On the other hand if the staining is too faint it requires more effort by the user to detect a &#8220;true&#8221; cell outline as thin cell protrusions can be missed.
However, by applying a fixed protocol of cell staining and array imaging procedure the range for this error becomes acceptably low. During this work different cell staining procedures were tested, based on various concentrations of crystal violet, the presence of surfactants like Tween 20 or Triton-X, or a combination of dyes by adding Eosin. Nonetheless, the intensity of the staining as well as its stability varied substantially from experiment to experiment. Only the use of Brilliant Blue G as described above showed an acceptable cell staining robustness combined with enough intensity to still see the thin cell outline but without interfering with the developed contour approach.
In general, there are options to modify the presented technique. For example by combining the bright-field techniques presented here with fluorescence microscopy staining organelles like the nucleus or the actin filament network, which can provide additional information about the underlying cell processes in context of cell mechanics. Furthermore, using long-chain dialkyl carboxycyanines (DiI or DiO) the microposts become fluorescent.14 Nonetheless, it is important to control each deviation from the presented technique. The supplementary Figures 10 and Figure 11 illustrate potential pitfalls. Images taken of a Coomassie Blue G stained NIH 3T3 fibroblast cell in the green fluorescence channel (Figure 10B and 10C) using the inverted microscope present the main micropost bodies but not their tips leading to a faulty position analysis (Figure 10E). Despite the best effort to refocus after switching from bright-field mode (Figure 10A) the micropost tips cannot be imaged, which could be due to absorption of the fluorescence signal by the cell stain. This is in contrast to images taken with the yellow fluorescence channel of an upright microscope (Figure 11B-11F) wherein multiple crisp images can be obtain along the z-axis. Here the user needs to ensure that the image is taken with the focal plane at the very tip (Figure 11C) and not somewhere closer to the micropost bottom to avoid calling faulty deflection values.
It should be noted that there are also technical limitations for micropost assays. For instance, the accuracy of a detected deflection depends on the quality of the optical setup. Microscopes are often optimized for fluorescence imaging wherein the light sensitivity of the connected camera has a higher priority than a large number of pixels. Naturally, this leads to images with a larger pixel size. Furthermore, it should be noted that bright-field images cannot determine if cell protrusions reach the substrate bottom. However, the commercial micropost arrays were tested using confocal microscopy. It was found that cells seem to accept the binary coating that promotes only adhesion on the micropost tips and not for the rest of the micropost array. Another factor derives from the positional error of the micropost array, which has been shown to be 0.2 &#181;m. Together with the given spring constant of 2.8&#160;nN/&#181;m this leads to an error in force read-out of 0.6 nN. Another constraint for micropost assays derives from the fact that the applied forces from microposts shared by multiple cells cannot be determined. Therefore, only isolated single-cells can be analyzed. A specific limitation for the presented protocol derives from the correct application of the sensitive contour approach, which requires a certain amount of training for the user in order to gain reliable results. Furthermore, for a faster analysis the user should take images wherein the micropost array is aligned to form a horizontal or a vertical line along the edge of the observation field. Despite the fact that the software algorithm for finding microposts within a hexagonal grid geometry is not limited by a rotation angle the imaging of microposts more than 5&#176; off-axis make it difficult to crop to an image section without containing incomplete microposts, which then leads to a failing of the &#8220;Generate Grid&#8221; function. In such a case the user can use the integrated rotation function before starting to analyze the micropost positions.
The described method was mainly applied to characterize two bone cancer cell lines, a parental cell line named HuO9, and a derived highly metastatic line denominated M132. In this context over six hundred single-cell images were taken in bright field mode and analyzed using the MechProfiler software. The subsequent data mining revealed that the parental cell line HuO9 exerts slightly more force and typically covers more microposts per cell than cells from the metastatic cell line M132. However the cells were not synchronized and therefore in different stages of cell cycle at the moment of fixation, which likely contributed to the broad range of results. By taking live cell images over time periods of up to 24 hr, it was found that after spreading many cells remain at their initial position whereas others migrate, spread, disconnect and migrate again with different velocities (see supplementary videos). This indicates that the mechanical profile of a cell is often not constant and can vary greatly across a cell population or even within a single cell depending on its current activity. Another contributing factor could be that on a micropost array a cell is forced to perform discrete migration steps to reach another micropost, which is in contrast to classic TFM, where cells adhere to flat surfaces and can migrate with minute steps. However, in order to analyze these small steps it is necessary to take fluorescent images of focal adhesion points, which requires access to very advanced microscopes and highly elaborate image analysis software. Nonetheless, the distribution of mechanical profiles within larger populations is still often a defining characteristic of a cell line. Thus a high-throughput method that enables characterization of a large number of cells is essential. The manual imaging process and open-access software presented here harbors great potential for scaling to fully automatic microscope systems.
In summary a robust traction force microscopy approach is presented that is readily adoptable in a standard cell biology lab. The entire workflow from cell seeding to image analysis is simple and effective. While the protocols described here are fully functional, improvements are ongoing to adapt the analysis algorithms to be able to also handle micropost arrays with orthogonal layouts and developing procedures that simplify the analysis of image sequences from live cell imaging. In context with the results for bone cancer cell lines, the approaches described here can be used to screen cells from clinical biopsies in order to establish whether such assays may hold prognostic value, enabling clinicians to predict the metastatic potential of biopsied cells from bone cancer patients. Such an assay would influence the therapeutic strategies. Other potential applications relate to testing pharmaceutically active compounds on mechanically active cells like smooth muscle cells or cardiomyocytes, potentially to assist in establishing a drug&#8217;s efficacy or toxicity.

Mechano-sensitive cell-based assays allow for investigating adherent cells, with a broad range of applications that reflect the central role that mechanics can play in cell biology. These applications often focus on the underlying mechanisms that drive subcellular processes or whole-cell behavior. On the one hand, external environmental factors such as extra cellular matrix composition or matrix stiffness can dramatically affect the mechanical and biological response of a cell.1 The same can be observed after use of many classes of pharmaceutically active compounds, the effects of which are often characterized using cell culture models.2 On the other hand genotypic properties, such as those caused by spontaneous or experimentally induced genetic mutations, can induce marked changes in cell phenotype that are associated with alterations in the cytoskeleton structure and function.3 These examples are just a few of the many possible topics for which mechanical phenotyping of cells is relevant, and all of these have been usefully investigated with micropost arrays.
At time of this writing, approximately 200 articles have been published describing cell-micropost interaction. These works discuss theoretical aspects of micropost deflection principles as well as practical instructions on their manufacture. The first article describing the interaction of cells and flexible micropost arrays was published by Tan and colleagues in 2003.4 In contrast to classic traction force microscopy (TFM) where continuous soft substrates are used to estimate nanonewton-scale cell contractility, Tan et al. described a method using multiple closely spaced vertical beams made of silicone elastomer. The main advantages of this technique emerge from two major features. First in order to change the cell-apparent substrate stiffness one only needs to change the micropost dimensions while keeping the substrate composition otherwise constant and thus avoiding differences in surface topology and chemistry. Second microposts act like individual springs that can be discretely analyzed with force and spatial resolutions on the order of individual focal adhesions and can reduce the analytical challenges that are inherent to analogous analysis by standard TFM.
Today the range of applications for micropost arrays greatly exceeds just the mapping of forces for a few single cells. For example, Akiyama reports the use of an isolated dorsal vessel tissue from a moth caterpillar as an actuator for a micropost array, in order to develop an insect muscle-powered autonomous micro-robot.5
However, most published applications of microposts have focused on studies of medical conditions like infection or cancer. For instance, micropost arrays have been used to study the force generation of bundled type IV pili of Neisseria gonorrhoea colonies that is associated with signal cascades enhancing infection.6 Others have used microposts to study breast cancer cells treated with pharmaceutical compounds targeting the cytoskeleton.7
Deflection of a micropost is often described using classical beam theory for a cantilever with an end load assuming the cell attaches only to the very tip of the micropost. Here the applied force F that causes a deflection &#948; depends on the micropost&#8217;s &#8220;bending stiffness&#8221; k and is calculated by:
<img alt="Equation 1" src="/files/ftp_upload/53350/53350eq1.jpg" /> (1)
with E, I, and L being the Young&#8217;s modulus, area moment of inertia and beam length respectively. However, results from this equation only give a general approximation of the forces at work since beam shearing and bending as well as substrate warping are not taken into account. Considering that microposts are typically made from soft materials like polydimethylsiloxane (PDMS)-based silicone rubber these factors need to be included. Schoen et al. demonstrated that there is such a correction factor based on the aspect ratio of the micropost (L/D) and the corresponding polymer&#8217;s Poisson ratio v.8 It is given by:
<img alt="Equation 2" src="/files/ftp_upload/53350/53350eq2.jpg" /> (2)
With Ttilt(v) being a tilting coefficient that includes fitting parameter a = 1.3 as can be found in the same article:
<img alt="Equation 3" src="/files/ftp_upload/53350/53350eq3.jpg" /> (3)
That means a micropost&#8217;s corrected stiffness kcorr is the product of the pure bending stiffness k=kbend and the correction factor corr given by:
<img alt="Equation 4" src="/files/ftp_upload/53350/53350eq4.jpg" /> (4)
Therefore, cell force calculations should be performed using the more refined variation of equation (1) now reading:
<img alt="Equation 5" src="/files/ftp_upload/53350/53350eq5.jpg" /> (5)
The impact of the correction becomes more obvious as soon as typical values for micropost dimensions are used. For example, a 15-micron long micropost with a circular cross section and a diameter of 5 &#181;m made of PDMS-based silicone rubber leads to a correction factor of 0.77 and therefore an uncorrected calculation would overestimate the exerted cell forces by 23%. This becomes even more severe for microposts with smaller aspect ratios.
Traditionally, micropost image analysis has also been based on idealized beam bending theory. In 2005 the group that pioneered the use of micropost arrays published an image analysis software suited for micropost analysis.9 The software requires a software license and the user must take three images for each position; one each from the micropost&#8217;s top and bottom planes in transmission mode and another one in fluorescence mode with the stained cell. After comparing the top and bottom positions for each micropost the software determines a force vector field and calculates related parameters like force per post. Other software packages exist and their analysis principles are briefly mentioned in the corresponding articles that describe them, but these analytical software packages are generally not publicly available.10,11
The micropost arrays designed for mapping cell forces can be classified as either being in an orthogonal micropost layout or a hexagonal one, the latter of which have the advantage of equidistant gaps between all neighbor microposts. Typical microposts have a circular cross section and their dimensions range from 1.0 &#181;m to 10&#160;&#181;m in diameter and 2 to 50 &#181;m in length. 4 However, microposts with elliptic or square cross section have also been reported.12,13
The use of PDMS-based silicone mixtures as micropost material allows for adding nanoparticles into the mixture. For example adding cobalt nano-rods enables a magnetic activation of the micropost and thus gives another degree of freedom to potential experimental designs.14 Most groups produce their micropost arrays on flat rigid substrates like cover glass or inside a Petri dish. However, Mann and co-workers recently reported a micropost array formed on a stretchable membrane.15 This allows the application of cell stretching forces to adherent cells while studying live-cell subcellular dynamic responses in terms of cell contractility.
The widely employed and most established process for making micropost arrays is based on soft lithography as described in the insightful protocols of Sniadecki and colleagues.16-18 In short standard cleanroom processes are used to generate the microstructures on top of a silicon wafer using SU8 photoresist. This is followed by a copying process wherein the silicone rubber is cast over the structures transferring them into molds. In a second step these molds are used to replicate the initial microstructure using silicone rubber on top of a chosen substrate. However despite the large and growing number of publications related to their application, establishing a manufacturing process for microposts takes considerable amount of time even for micro-engineering experts; there are many process steps that require optimization and adaptation to the specific lab environment and micropost layout to yield an acceptable quality level.
Commercial micropost arrays are now available in a ready-to-use (&#8220;off-the-shelf&#8221;) format with a consistently high quality. As such they are an alternative to the complex and lengthy manufacturing process required for on-site production. In this paper a commercially available micropost array was used for mapping cellular forces using a single bright-field microscopy image. More importantly this article describes and documents a fully-functional open-source software named MechProfiler, which is available for download as supplementary material to this manuscript. An actively maintained version of the software can also be found at http://www.orthobiomech.ethz.ch.
The combination of an &#8220;off-the-shelf&#8221; assay and a compatible open-source analysis software markedly lowers the entry hurdle to achieve accurate TFM experiments. Researchers without access to either clean room facilities or software development expertise can analyze cellular forces successfully. It enables a user to focus on the mechanosensitivity assay output rather than the technology itself, and makes traction force measurements available to a broader community. Furthermore, this is an important step to pave the way towards fully automatic screening of micropost arrays.
The MechProfiler analysis software processes images in file format tiff, png, bmp and jpg. The images can be taken using fluorescence, phase contrast or bright-field light microscopy. The standalone program runs together with the free Matlab Compiler Runtime (available at: Figure 12) and underlying algorithms allow for streamlined image processing, which enables the user to process images with single or multiple cells in about 1 min. Further, these cells may either be living or &#8220;fixed&#8221;.
The MechProfiler software is able to greatly increase data analysis throughput by relying on reproducibility of quality commercial micropost arrays, more specifically, the default &#8220;non-deflected&#8221; position of each post in the array can be presumed against an ideal grid (manufacturing deviations for the grid in the arrays used for this study were less than 100 nm).
In short one opens a selection of image files for analysis, crops them to the region of interest, defines the posts covered by cells or which need to be discarded, determines the post positions, calculates the deflections/forces against the ideal grid, and finally saves all cell-specific data with a possibility for export, including to a standard office spreadsheet.

<table><tbody><tr><td>T25 cell culture flasks</td><td>Nunc</td><td>156367</td><td></td></tr><tr><td>1x PBS-buffer</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>0.5% Trypsin-EDTA</td><td>Life Technologies</td><td>15400054</td><td></td></tr><tr><td>Medium DMEM/F12</td><td>Sigma</td><td>D8437</td><td></td></tr><tr><td>FBS South America</td><td>Life Technologies</td><td>10270106</td><td></td></tr><tr><td>Penicillin-Streptomycin 100x</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>15 ml centrifuge vials</td><td>Sarstedt</td><td>62.554.502&amp;nbsp;</td><td></td></tr><tr><td>Micropost array pre-coated with Collagen I/Fibronectin</td><td>MicroDuits</td><td>MPA-col1/FN</td><td>Micropost dimensions: &amp;Oslash;=6.4&amp;#160;&amp;micro;m, l=18.2 &amp;micro;m; grid=13 &amp;micro;m, k&lt;sub&gt;corr&lt;/sub&gt;=2.87&amp;#160;nN/&amp;micro;m</td></tr><tr><td>Ethanol abs p.A.</td><td>Merck</td><td>100.983</td><td></td></tr><tr><td>12-well plate</td><td>Nunc</td><td>150628</td><td></td></tr><tr><td>Formalin solution, neutral buffered 10%</td><td>Sigma</td><td>HT5011</td><td></td></tr><tr><td>Brilliant Blue G-250</td><td>Sigma</td><td>27815</td><td>Coomassie blue</td></tr><tr><td>Methanol ACS p.A.</td><td>Merck</td><td>1.06009</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td>695092</td><td></td></tr><tr><td>Glass bottom dish</td><td>WillCo Wells BV</td><td>GWSb-3522</td><td>35 mm diameter, aperture 22&amp;nbsp;mm</td></tr><tr><td>T-100 Eclipse</td><td>Nikon</td><td>n/a</td><td>Inverted microscope</td></tr><tr><td>D3-L3</td><td>Nikon</td><td>n/a</td><td>Camera controler</td></tr><tr><td>DS Fi2</td><td>Nikon</td><td></td><td>Camera</td></tr></tbody></table>

easy and accurate mechano-profiling on micropost arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Watch this Scientific Journal Video about Easy and Accurate Mechano-profiling on Micropost Arrays at JoVE.com

Easy and Accurate Mechano-profiling on Micropost Arrays

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

easy-and-accurate-mechano-profiling-on-micropost-arrays

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.1K Views.  Balgrist University Hospital. Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost. 

Video: Easy and Accurate Mechano-profiling on Micropost Arrays

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Biology

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Parallel Active Cantilever Arrays in AFMS to Enable High-Throughput Inspections

Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays for High-Throughput Large-Scale Sample Inspection

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. However, due to their limited imaging throughput, AFMs have not been widely adopted for large-scale inspection purposes. Researchers have developed high-speed AFM systems to record dynamic process videos in chemical and biological reactions at tens of frames per second, at the cost of a small imaging area of up to several square micrometers. In contrast, inspecting large-scale nanofabricated structures, such as semiconductor wafers, requires nanoscale spatial resolution imaging of a static sample over hundreds of square centimeters with high productivity. Conventional AFMs use a single passive cantilever probe with an optical beam deflection system, which can only collect one pixel at a time during AFM imaging, resulting in low imaging throughput. This work utilizes an array of active cantilevers with embedded piezoresistive sensors and thermomechanical actuators, which allows simultaneous multi-cantilever operation in parallel operation for increased imaging throughput. When combined with large-range nano-positioners and proper control algorithms, each cantilever can be individually controlled to capture multiple AFM images. With data-driven post-processing algorithms, the images can be stitched together, and defect detection can be performed by comparing them to the desired geometry. This paper introduces principles of the custom AFM using the active cantilever arrays, followed by a discussion on practical experiment considerations for inspection applications. Selected example images of silicon calibration grating, highly-oriented pyrolytic graphite, and extreme ultraviolet lithography masks are captured using an array of four active cantilevers (&#34;Quattro&#34;) with a 125 &#181;m tip separation distance. With more engineering integration, this high-throughput, large-scale imaging tool can provide 3D metrological data for extreme ultraviolet (EUV) masks, chemical mechanical planarization (CMP) inspection, failure analysis, displays, thin-film step measurements, roughness measurement dies, and laser-engraved dry gas seal grooves.

Author Spotlight: Introduction to Active Probe Atomic Force Microscopy with Quattro-Parallel Cantilever Arrays

Large-scale sample inspection with nanoscale resolution has a wide range of applications, especially for nanofabricated semiconductor wafers. Atomic force microscopes can be a great tool for this purpose, but are limited by their imaging speed. This work utilizes parallel active cantilever arrays in AFMs to enable high-throughput and large-scale inspections.

An Atomic Force Microscope&#160;(AFM) is&#160;a powerful and versatile tool for nanoscale surface studies to capture 3D topography images of samples. ...

Engineering

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay.  Automated qPCR setup has improved this field by allowing for greater reproducibility.  Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment.  This method along with internal plate controls also reduces experimental variables common to other techniques.  
We recently developed a qPCR assay for profiling of pre-microRNAs  (pre-miRNAs) using a set of 186 primer pairs.  MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level.  These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA).  Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs.  Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful.  There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique.  Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs.  Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression.  However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing.  Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Mechano-Node-Pore Sensing: A Rapid, Label-Free Platform for Multi-Parameter Single-Cell Viscoelastic Measurements

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer metastasis. Conventional methods for measuring these properties, such as atomic force microscopy (AFM) and micropipette aspiration (MA), capture rich information, reflecting a cell's full viscoelastic response; however, these methods are limited by very low throughput. High-throughput approaches, such as real-time deformability cytometry (RT-DC), can only measure limited mechanical information, as they are often restricted to single-parameter readouts that only reflect a cell's elastic properties. In contrast to these methods, mechano-node-pore sensing (mechano-NPS) is a flexible, label-free microfluidic platform that bridges the gap in achieving multi-parameter viscoelastic measurements of a cell with moderate throughput. A direct current (DC) measurement is used to monitor cells as they transit a microfluidic channel, tracking their size and velocity before, during, and after they are forced through a narrow constriction. This information (i.e., size and velocity) is used to quantify each cell's transverse deformation, resistance to deformation, and recovery from deformation. In general, this electronics-based microfluidic platform provides multiple viscoelastic cell properties, and thus a more complete picture of a cell's mechanical state. Because it requires minimal sample preparation, utilizes a straightforward electronic measurement (in contrast to a high-speed camera), and takes advantage of standard soft lithography fabrication, the implementation of this platform is simple, accessible, and adaptable to downstream analysis. This platform's flexibility, utility, and sensitivity have provided unique mechanical information on a diverse range of cells, with the potential for many more applications in basic science and clinical diagnostics.

Presented here is a method to mechanically phenotype single cells using an electronics-based microfluidic platform called mechano-node-pore sensing (mechano-NPS). This platform maintains moderate throughput of 1-10 cells/s while measuring both the elastic and viscous biophysical properties of cells.

Cellular mechanical properties are involved in a wide variety of biological processes and diseases, ranging from stem cell differentiation to cancer ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...

Easy and Accurate Mechano-profiling on Micropost Arrays

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