I'm assuming you culture cells on a fluorescent-tagged gelatin layer and image the plates for invadopodia degradation. If so, you need to open the microscope files in FIJI (imageJ would have a rough time opening raw microscope files). Then you need to use "Auto local threshold" tool in order to threshold for ECM degradations. Go to Image>Adjust>Auto Local Threshold. Auto local threshold menu pops up. Let the method be "Try all" and click OK. The local threshold will process your image and threshold it with all the predefined algorithms. Results will be a mosaic image containing the results of all the algorithms. Here you need to look at your actual image of the gelatin and compare it with the thresholding result. Any of the thresholding results that is closest to what you want would be the algorithm that you wanna use. Once you selected your algorithm, go to Image>Adjust>Auto Local Threshold. This time from select the name of the algorithm that worked the best for you from the drop down menu and click ok. You can further improve the results by playing around with the "Special parameters" of the Auto Local Threshold menu.

Once you acquired the thresholded, binary image of degradation, You need to count the number of degradations and measure the area of each degradation spot. Click Analyze>Analyze Particle. From the drop down menu select "Overlay" and click OK. All the particles and their areas will be counted and the number and their sized will be reported to you. If you have a little bit of noise being thresholded as false positives, you can play around with the adjustments of the particle analysis tool such as circularity range and size range in order to refine the algorithm.

Take a look at my recent paper:

http://www.cell.com/biophysj/fulltext/S0006-3495(18)30186-3

I have explained these in the method sections of my paper as well.

Usually in invadopodia formation assay we evaluate the invadopodia number and their length, also calculate how many invadopodia have migrating cells. Those processes are related to gelatin degradation.

I'm assuming you culture cells on a fluorescent-tagged gelatin layer and image the plates for invadopodia degradation. If so, you need to open the microscope files in FIJI (imageJ would have a rough time opening raw microscope files). Then you need to use "Auto local threshold" tool in order to threshold for ECM degradations. Go to Image>Adjust>Auto Local Threshold. Auto local threshold menu pops up. Let the method be "Try all" and click OK. The local threshold will process your image and threshold it with all the predefined algorithms. Results will be a mosaic image containing the results of all the algorithms. Here you need to look at your actual image of the gelatin and compare it with the thresholding result. Any of the thresholding results that is closest to what you want would be the algorithm that you wanna use. Once you selected your algorithm, go to Image>Adjust>Auto Local Threshold. This time from select the name of the algorithm that worked the best for you from the drop down menu and click ok. You can further improve the results by playing around with the "Special parameters" of the Auto Local Threshold menu.

Once you acquired the thresholded, binary image of degradation, You need to count the number of degradations and measure the area of each degradation spot. Click Analyze>Analyze Particle. From the drop down menu select "Overlay" and click OK. All the particles and their areas will be counted and the number and their sized will be reported to you. If you have a little bit of noise being thresholded as false positives, you can play around with the adjustments of the particle analysis tool such as circularity range and size range in order to refine the algorithm.

Take a look at my recent paper:

http://www.cell.com/biophysj/fulltext/S0006-3495(18)30186-3

I have explained these in the method sections of my paper as well.

Thank you for valuable comment.