available at the
bottom of the page.
If you encounter any problems or find any of these instructions
confusing, feel free to email me (matthew.berginski [AT]
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.
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.
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
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
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:
Now suppose you want to set the default time between images to
0.5 and minimum adhesion size to 5 pixels:
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:
to the submission form.