DynaCyte 1.0. Operations Manual

Transcription

1 DynaCyte 1.0 Operations Manual Manuel Gómez, Baldomero Alonso Latorre, Ruedi Meili, Katie Osterday, Juan C. del Álamo, Richard A. Firtel and Juan C. Lasheras September 2009 Laboratory of Bio-Fluid Mechanics Department of Mechanical and Aerospace Engineering University of California, San Diego La Jolla, CA

2 Software Installation and Setup Introduction DynaCyte 1.0 is a graphical interface program specifically designed to calculate the 2D shear stresses exerted on the surface of a flat elastic substrate given a map of shear deformations measured at an arbitrary plane parallel to the surface. These deformations can be obtained by tracking the displacements of beads embedded in the substrate while the stresses are applied. DynaCyte 1.0 has been specifically designed to track the forces exerted by a cell during its motion over a flat surface. In order to achieve this goal, the user must provide the deformations of the surface and the substrate parameters, and DynaCyte 1.0 will return a map of the stresses over the surface. An image of the cell is used for graphical representation purposes but is not used in the calculations (this can be easily checked in DynaCyte 1.0 by introducing a random image as cell image). DynaCyte 1.0 makes use of the improved Fourier traction cytometry algorithms described by del Álamo et al. (2007) in PNAS 104 no. 33, Manuel Gómez has developed the user interface using Visual Basic. If you have any question or suggestion regarding DynaCyte 1.0, or you would like to receive notifications and updates about future versions of DynaCyte, please send an to Manuel Gómez, at manu@ucsd.edu. Installation Two files are necessary for installing and running DynaCyte 1.0 on MS Windows XP and Vista: MCRInstaller.exe: the Matlab compiler, necessary to run the Matlab code embedded in DynaCyte. DynaCyte_1.0.zip: a zip file. Both of these files can be found on the webpage: It is necessary to install MCRInstaller.exe first. After the installation of MCRInstaller.exe is complete, unzip the file, DynaCyte_1.0.zip, and copy all of its content into any folder. To execute the program, double-click on DynaCyte.exe. Examples Examples can be found in the folder titled: Examples. This folder is contained in the DynaCyte_1.0.zip file and is not required by the program to run. It can be deleted freely in case the user needs to free up hard disk space.

3 Basic Software Operation DynaCyte 1.0 operates through six consecutive steps. Step 1: Select the Cell Image User-Interface Overview When DynaCyte.exe is executed, a window, like Figure 1, is prompted. This window has four main areas. Directions on how to fulfill each step of the program are given in the header located in the top left corner. At left, there are two boxes: the directory browse on top, and the file search below. At right, there is a preview box, which displays the selected files, and below it are the control buttons. Figure 1: First window of DyneCell.exe Uploading an Image File In order to begin processing an image file, the image directory must be selected. Select the target folder or disk drive by single-clicking the icon in the directory browse. Permissions Error In some systems, there could be a problem with file searching caused by not having adequate file-access permissions. If your user does not have permissions to open the image folder in your system, an error message will occur. This problem is most likely to arise when attempting to access the desktop or home folder, because not every user has access

4 permissions for the Documents and Settings folder in Windows. If this error occurs, go to the directory: C:\Users\ and select your user name. From this directory, you will be able to access the desktop. Once the image folder is selected, the user needs to select the image file, by clicking on the desired image in the file search box. Once the correct image is properly identified, a preview of the image will be displayed in the preview box, as depicted in Figure 2. Figure 2: Select the image file. Warning: TIFF Files with over-8-bit color depth. Please Note: DynaCyte 1.0 can process TIFF images with over-8-bit color depth, but will not display them in the preview box due to current limitations of Windows Preview. This minor issue does not affect the calculations and will be addressed in future versions of the program. To continue, click Next. Making Revisions From this point on, clicking the Previous button will allow the user to review or change any of the previous settings. Step 2: Select the Deformations File User-Interface Overview Once the Next button is pressed, a similar window will be displayed, as shown in Figure 3. This window contains a file search box that displays.txt files and a preview box that

5 displays the text contained in the.txt file, rather than the preview image previously displayed. Figure 3: Search the input file screen. Data Storage: Input File The input file provided by the user, must be a text file containing the coordinates of the measured displacements of the embedded beads; stored in four columns. The deformations must be measured along a single plane using a Cartesian grid. DynaCyte 1.0 will use these deformations to calculate the forces exerted on the substrate. In order to obtain the deformations, the user can employ any Particle Image Velocimetry or Particle Tracking software, such as GPIV, OpenPIV, or any other commercial software. Ordering Input Points DynaCyte 1.0 interprets the deformations as follows: The first column corresponds to the x coordinates of the points where the deformations have been measured, measured in pixels. The second column corresponds to the y coordinates of the points where the deformations have been measured, measured in pixels. The third column corresponds to the shear deformation, ε xz, measured in pixels. The fourth column corresponds to the shear deformation, ε yz, measured in pixels. The coordinates must also be ordered from the bottom row upwards, beginning on the lefthand side; ending at the upper right-hand corner. If, in the deformations measurement process, the user obtains an input file arranged in a different order, the deformations map will not match the input image. This should be obvious to the user when both image and deformations are displayed overlapped in the fifth step.

6 If the deformations file does not contain four columns, an error will occur, and the user must either select another file, or fix the number of columns contained in the.txt file. The user must ensure that each column in the input file has the data stored in the correct order; otherwise the results will be incorrect. Select the input file By default, the previously selected folder, the cell image directory, is selected, but the user can browse and select the folder where the deformations file is located. After the input file is properly selected, the columns described above are displayed in the preview window, as shown in Figure 4. Press Next to continue. Figure 4: Select the input file. Step 3: Select the Output File User-Interface Overview Once the Next button is pressed, the preview-text window clears, as shown in Figure 5. In this step, the user must specify the output file where DynaCyte 1.0 will store the calculated deformations. Force Calculations: Output File The output file is a text file where DynaCyte 1.0 stores the calculated forces. The output data is stored in the following order (similar to the input data): The first column corresponds to the x coordinates of the points where the stresses are

7 calculated The second column corresponds to the y coordinates of the points where the stresses are calculated The third column corresponds to the shear stress, τ xz. The fourth column corresponds to the shear stress, τ yz. Ordering Output Points DynaCyte 1.0 sorts the output data in the same way as the input data. The points are ordered from the bottom row upwards, beginning on the left-hand side; ending at the upper righthand corner. Select or Create Output File Figure 5: Select the output file. To select an output file, follow the same steps previously described in Step 1: Select a Cell Image. To create a new output file, type the desired name in the text box, and click the Create file button. Once the output file is selected, press the Next button to continue. Step 4: Defining Experimental Parameters User-Interface Overview Next, the user will be prompted to define the experimental parameters required to obtain the traction shear stresses from the input file, as shown in Figure 6.

8 Figure 6: Parameters Screen The properties needing to be defined include: Elastic and Young's Modulus, in Pascals and adimensional, respectively. Geometrical parameters such as: o Substrate thickness, in microns. o Distance of the measurement plane to the surface of the substrate, in microns. Calibration factor of the image, with units of micrometers per pixel of the image. This is the size, in microns, of each image pixel. To continue, click Calculate. Step 5: Select Region User-Interface Overview Once the Calculate button is pressed, the image and the deformations map are displayed, as shown in Figure 7.

9 Figure 7: Deformations map. Select Region After the deformations map is displayed, the user must select the region of the image where he/she would like to calculate traction forces. To select a region, click on a target spot on the image and drag the mouse. A rectangle will highlight the area of interest, as shown in Figure 8. It is possible to select the whole image. Figure 8: Area to calculate the forces.

10 Step 6: Access Force Calculations After the region of interest is selected, a new window appears, containing a map of the forces, as shown in Figure 10. Figure 2: Map of the forces. The numerical values of the forces are stored in the selected output file for future reference.