.AVI file

[Mateus Borges]

Thank for the package! I'm working with bull sperm, but I can't open the video file (the video format is .avi). The progress bar on ImageJ doesn't go untill the half, and it stops. Any instruction for the best use? Is it correlated with the bits rate of the video?

[Carlos Alquezar]

Hi Mateus!

Have you tried with the sample AVI files available in https://figshare.com/articles/OpenCASA_dataset/7247039/1? If not, please try first with these files. If it doesn't work, I could mean that the plugin is not compatible with your ImageJ version or your OS. In the case that it works, it could mean that the problem is in your AVI files.

[Felipe Martínez-Pastor]

Hi, what system are you using? Maybe MacOS like me?

For me, it works fine in Windows 10, but not in MacOS X. Here is my setup:

macOS Mojave (10.14.3)

iMac 2017 and MacBook Pro 2017, 16 and 8 GB RAM

ImageJ 1.52k, last OpenCASA installed.

I have only tried the motility module.

The plugin installs and starts with no problem. When I select Motility/open file, it shows nothing and I cannot interact with ImageJ anymore. Not hanged or frozen, it's like the focus is in a hidden window. I have to force quit.

When I select Directory, it shows file browser window (strangely, it sometimes work with the "File" option, but only in the MacBook). However, it does not seem to process the videos (with File, it shows activity and the stops, with Directory, it just shows blank "Individual motility" and "Average motility" windows).

The videos can be opened in ImageJ and analyzed with the Wilson-Leedy/Purchase plugins.

Greetings!

Felipe

El jueves, 28 de febrero de 2019, 13:22:05 (UTC+1), Mateus Borges escribió:

[Mateus Borges]

Hi, I'm using Windows 10. I've tried the motility module, but I still can interact with the software. The only problem is with the load of the file.

[Carlos Alquezar]

For the moment, as it is said in the paper, the plugin has been validated only on Windows 7 (64-bit) using ImageJ v1.49q and Java JRE 1.8.0_101 (64-bit). For some reason, the plugin (in particular, the .jar generated on windows), does not work properly on MacOS. This is something that I hope it will be fixed in the future, but for the moment any feedback is well received to take it into account.

[Felipe Martínez-Pastor]

Just tell me if you need a beta-tester! Good work!

By now, I'll work with Win10, I'll give a try in Linux if I have the opportunity.

[Carlos Alquezar]

Thanks for the offer!! Good to know :) If I try to test and fix it on MacOS I will contact you for sure

[Yuansheng Tan]

What I found out it that OpenCASA only works with AVI files with the following encoding properties:

Format                                   : RGB

Codec ID                                 : 0x00000000

Codec ID/Info                            : Basic Windows bitmap format. 1, 4 and 8 bpp versions are palettised. 16, 24 and 32bpp contain raw RGB samples

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[Mateus Borges]

Thank you all for the help! I've made it run by saving the original .avi file with ImageJ. But I have another question. What technique was used for the sample images? I'm using a Leica bright-field microscope, making the spermatozoa cells do not appear correctly. 

Regrets!

[Carlos Alquezar]

Hi Mateus!

let me if I am wrong but I think that the information you are asking for is in the motility validation section in https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1006691.

For the video recordings, it was not included in this work but we also tried a basler camera with the Pylon software and the program worked well with those videos.

[Mateus Borges]

Hi Carlos!

I was looking for the motility section, thanks! But my another question was about the technique used to make the images. Was used some kind  of staining or just observed the spermatozoa in a covered glass slide? I'm asking this because in the sample videos the background is black, and the spermatozoa are white. I am also having some trouble with the OpenCASA because theplugin is recognizing the cells onlly in the corners of the video. Is there something that I can do to resolve this?

Thanks!

[Carlos Alquezar]

Hi Mateus!

The videos were recorded using negative phase contrast in the microscope. This is the reason why the background looks black and the cells are white.

About your detection problem, have you changed the default settings?

[Mateus Borges]

Thanks for the help! We adjust the settings about cell size, as we measured with the microsccope software.

[Carlos Alquezar]

Perfect. Be sure also that the microns per pixel parameter coresponds with your setup. Let me know if you could analyze the sample videos with the default parameters, or you found the same problems detecting the cells only in the corners. If you could analyze them, but your recordings are not made by a negative phase contrast, the problem could be there.

[Michael Botts]

Hi Tony et al.,

This Codec info was super helpful to convert video files to the correct AVI for OpenCASA (I am using on Windows 10). I have videos that were in the .mp4 format and I converted to .avi using ffmpeg using ffmpeg: ffmpeg -i input.mp4 -vcodec rawvideo -pix_fmt bgr24 ouput.avi. 

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[Felipe Martínez Pastor]

Very interesting information about the AVI format and the use of ffmpeg
(especially considering massive conversion via scripts). There is plenty
of video software and sometimes they don't like writing files in the
format you need!


El 26/3/19 a las 18:13, Michael Botts escribió:

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[Tan]

That was what I used for encoding! Use gray instead of rgb24 to
reduce size to one third of the size.

Tony

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[Michael Botts]

I am still new to ffmpeg. Using gray instead of rgb makes a ton of sense. I will likely be doing that in the future also. 

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[Erick J R Silva]

Hi Tony,

Thanks for the tip!

Could you please provide the command line to have the videos converted in gray scale rather than rgb24 via ffmpeg?

Kind regards,

Erick

Em terça-feira, 26 de março de 2019 14:31:10 UTC-3, Tony Tan escreveu:

That was what I used for encoding!   Use gray instead of rgb24 to
reduce size to one third of the size.

Tony

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[lindsayspen]

Hi Mateus, 

I'm having a similar problem. Only some of my .AVI files will work in the motility module. What process did you use to make them work? I have tried saving as .AVI in imageJ to no avail.

Thanks, Lindsay

[lindsayspen]

It also seems that files won't work unless they are in an external drive (ie. shared drive or USB). No idea why this is, anyone go some insight?