Instructions
Thank you for helping to test the invadopodia analysis webserver. If you
encounter any problems, feel free to email me (matthew.berginski [AT]
gmail.com).
Required Files
All files should be submitted as raw-data grayscale TIFF files.
The processing pipeline also expects that the files submitted
were taken simultaneously or as close as is possible with your
imaging setup. Thus, the ECM imaged in the first frame of the
time-lapse should be from the same region and time as the first
frame of the puncta time-lapse.
Puncta Time-lapse
The puncta file should look something like the sample file
linked from the home page. Namely, the bright regions of image
should correspond to the potential invadopodia. The system has
been tested with Lifeact-GFP images, but I expect that images of
other invadopodia componenets will also be acceptable for
processing.
ECM Time-lapse
The puncta file should look something like the sample file
linked from the home page. Namely, the field of view should be
uniformly bright, with the regions corresponding to invadopodia
degradation appearing dim compared to the surronding area. The
processing pipeline can deal with some variation in the
uniformity of the labeled ECM.
Experiment Configuration
Median Filter:The cell outlines are idenfied from the
puncta images, which occassionally require a median filtering
step. Select this option if the cells in your images are not
being detected.
Puncta Seed and Expansion Thresholds:Puncta
identification uses two thresholds to determine where the puncta
are located in each image. First a set of seeds pixels are
identifed using the seed threshold and then those seeds are
expanded based on the expansion threshold. You also have the
option to eliminate seeds below a certain size using the puncta
seed minimum size settings.
If you want to see what one of your images looks like when
processed with a specific threshold try out the threshold tester
available through the Focal Adhesion Analysis Server. The
algorithm used to idenfity adhesions is very similar to the one
used to identify the puncta, so the results from this service
are a good guide as to how you should set these parameters.
Min/Max Size: These settings control the
minimum or maximum puncta sizes allowed through the
segmentation step, both are specified in pixels.
Max Major/Minor Axis Ratio: This setting controls
maximum allowable ratio between the major and minor axes of an
ellipse fit to a potential puncta. This filter allows very
elongated structures to be automatically removed from
consideration.
ECM Normalization
This value sets the average intensity of the ECM outside the
cell bodies in your ECM images. Setting this value will allow
comparisons to be made between the intensities of seperate
fluorescent ECM preparations. You can remove the setting by
leaving the box blank. When blank, the ECM will be photobleach
corrected to the first image in the image set.
Optional
ECM Flat Field Correction File
If this image is submitted, it will be used to flat field
correct the ECM image set.
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.