Full instructions available at the bottom of the page.

Required File

TIFF formatted time-lapse image set of the focal adhesions

Experiment Configuration

Amount of time, in minutes, between the images

Segmentation Options

If selected, the watershed segmentation methods are deactivated
If selected, a median filter will be applied before segmentation
Normally set to 2, see instructions below for more help

Adhesion Size Options

Leave blank to remove the minimum adhesion size limit
Leave blank to remove the maximum adhesion size limit

Analysis Options

The minimum number of points in an assembly or disassembly phase

FAAI Options

The minimum value of the FA major/minor axis for inclusion in the FAAI

Optional Cell Mask Image Set

TIFF formatted image to used to find the edge of the cell

Notification Options

Your email address will only be used for notification purposes
Any text in this box will be returned with the results email

Instructions

If you encounter any problems or find any of these instructions confusing, feel free to email me (matthew.berginski [AT] gmail.com).

Required Files

Adhesion Image File

The program expects that you will submit a grayscale stacked TIFF image set of the Focal Adhesion marker protein. The analysis methods have been tested with a wide range of Focal Adhesion markers including Paxillin, FAK and Vinculin. The image set can be of any size, but keep in mind that a large image set can take some time to upload.

Detection Settings

Adhesion Detection Threshold:This number is used by the adhesion detection script to determine when a pixel is or is not part of an adhesion. After appling a high pass filter to the images, pixels above this level are considered part of an adhesion, while the pixels below are classified as background. The lower this number, the more pixels will be classified as part of an adhesion. The default value of two works well in most cases, but values down to around one may be appropriate. Also be aware that lower values will lengthen the runtime. If you want to see what one of your images looks like when processed with a specific threshold try out the threshold tester.

Apply Median Filter: This option applies a 7 by 7 median filter to the focal adhesion image during segmentation. I've found this setting useful for segmenting adhesions imaged using confocal microscopy. The filtered image is only used for segmentation purposes, so all downstream properties are based on the raw image values. If using this setting, I also suggest you select the "Don't Split Adhesions Option", otherwise the watershed segmentation has a tendency to oversegment the FAs.

Don't Split Adjacent Adhesions: This option turns off the wateshed segmentation method normally used to split adjacent adhesions. The watershed segmentation is the slowest part of the image processing, if this option is selected, processing runtimes decrease. You might want to turn on this option if the adhesions in your experiments don't appear to touch one another.

Min/Max Adhesion Size: These settings control the minimum or maximum adhesion sizes allowed through the segmentation step, both are specified in pixels.

Analysis Options

Only Calculate Static Properties: This option turns off the all the processing steps after calculating the FA properties for each image. None of the adhesions are tracked and none of the dynamic properties are calculated.

Min FA Phase Length: When determining the assembly and disassembly rates of the FAs in the image set, the analysis methods use log-linear models to determine the value of the rates. This setting specifies the minimum number of data points that will be included in the assembly and disassembly phases. Decreasing this value allows more assembly and disassembly phases to be gathered, but at the cost of those phases to potentially be built on fewer data points.

Min FAAI Ratio: The first step in calculating the FAAI is to filter FA's with a low major/minor axis ratio. The angle of a single FA can't be reliably determined when this ratio is low. The default value of 3 has worked for the image sets in the Cell publication (see front page), but the adhesions in your experiments may need a different value.

Additional Image Sets

Cell Mask: If a set of images are specified as the cell mask set, the software will attempt to find the cell body from these images. The methods work well in cases where the entire body of the cell is expressing a marker and a substantial portion of the background is also visible. If such a set of images are provided, various properties concerning the adhesions and their distance from the cell edge and cell centroid will be calculated.

Notification Options

Email Address

If an email address is provided, you will be notified via email when your job finishes processing. Your email address will only be used for notification purposes. If this is not provided, then the experiment status page returned on submission needs to bookmarked in order to retrieve your results.

Note to Self About Experiment

Whatever you put in this box will be send back to you in any email the system sends concerning your experiment. It is limited to 80 characters.

Changing the Default Values from the URL Bar

You can change the default values for all the experimental parameters by appending a few characters to the upload URL. The general format for this is ?VARIABLE_NAME1=VAL1&VARIABLE_NAME2=VAL2. For example, if you want to set the default time between images to 0.5, you can use this URL for the upload page:

http://faas.bme.unc.edu/upload?time_spacing=0.5

Now suppose you want to set the default time between images to 0.5 and minimum adhesion size to 5 pixels:

http://faas.bme.unc.edu/upload?time_spacing=0.5&min_adhesion_size=5

In order to set the checkboxes, such as enabling static analysis, using a value of 1 for on and 0 for off. The rest of the variable names should be self-explanitory:

  • stdev_thresh
  • static
  • no_ad_splitting
  • min_linear_model_length
  • min_adhesion_size
  • max_adhesion_size
  • FAAI_min_ratio
  • email
Return to the submission form.
Starting Upload

Monitoring your upload:

  • Google Chrome: In the lower left hand corner, you should see a percentage of how much of your upload has finished.
  • Firefox: In the tabs bar, there will be a rotating indicator and in the lower left hand corner a note about "Sending request to ..."
  • Internet Explorer: In the tabs bar, there will be a rotating indicator