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Hi Dongseok. Thresholding is subjective. There is no way we can say that a certain threshold level is the correct one. It will always depend on what you think is able to separate out your objects from the background as well as your own knowledge on what needs to be measured. The best way to do your analysis is to decide which automatic threshold algorithm to use, and stick with it for all your images. Thresholding is crucial if you want to isolate whatever you are measuring from the background.

@@E.Her100you can find this information in the 3D ROI Manager.

@@pierremangeol4387 thank you Pierre for a great toolset. I hope I was able to demonstrate what KymographClear can do, albeit a short overview.

@@johanna.m.dela-cruz Your presentation is perfect 🙂 I wish I did it when we published it. I try to do it on our new tools. Your format with text only comments works very well.

@@MM-v1k9f Tiff files are definitely compatible with Fiji. A number of file formats in microscopy, medical imaging, and related life sciences also work with Fiji because of the BioFormats plugin.

@@johanna.m.dela-cruz Thank you so much!

@@aleevareela hello! You can try either the local thickness plugin or BoneJ. Geodesic distance maps can also be helpful.

Hello @AbhishekBalmik-eu3nr. You basically should process your images the same way for both the control and positive cells. Setting a size range for puncta to be included is also a good idea...again, this setting should be the same for both kinds of images.

@AnduneYavetil, thanks for your support. This isn't something new...just wanted to make it easier for ImageJ/Fiji users to check out tools to do image analysis.

@@ibowman_UCLA_BRAIN Thanks for letting me help you.

@@selvamarin-k4o hello. When you acquired your images, did you have an overlap percentage for stitching?

@@johanna.m.dela-cruz To be honest, I don't know the exact overlap percentage of my pictures, but when I tried to set an overlap percentage and compute overlap , my pictures could not be sewn successfully, When I don't calculate the overlap rate, the pictures could be stitched together normally

@@johanna.m.dela-cruz I think the black lines between the pictures are caused by the uneven lighting of my pictures, but I don't know how to deal with this problem

@@selvamarin-k4o you could try applying a FFT bandpass filter. I have a tutorial on this in one of my videos: th-cam.com/video/SjwzCUYIFJ8/w-d-xo.html

@@johanna.m.dela-cruz thank you！

Hi there. I’m nit sure what you mean by not loading….do you have objects in the 3D Manager?

@@rajaaalwodai5745 hi! Thresholding works best when there is a good contrast between your object and the background. Perhaps your image needs a different method of segmentation. Have you tried weka segmentation or maybe Labkit?

@ Hi, Thanks for replaying. How do I do that? I’m trying to automate the segmentation and then measure the volume of the lymph nodes rather than manually segmenting each node. I’m doing this on an mri scan / a stack of images rather than an individual one. Can you please provide some assistance. Thanks again

@@rajaaalwodai5745 I do have tutorials on Weka Segmentation and LabKit. Try checking them out.

@@johanna.m.dela-cruz Ok that’s Just to clarify, do these segmentation processes work on a stack of images ?

@@rajaaalwodai5745 yes, they do.

@@claireseelingbranscomb2781 hi there. Do you have 2 channels and would like to count objects in one channel using ROIs from the other channel? If yes, then I would give your first question a thumbs up. The 3D viewer is just one way to view your image in 3D. You would first have to merge the channels in your images before you do 3D reconstruction.

@@johanna.m.dela-cruz Thank you for the reply. My biggest concern now is numbering the cells. Is there any way to add numbers to the 3D viewer?

@claireseelingbranscomb2781 I don't think the 3D viewer will show the numbers. You can try 3D Project or 3D Script.

@prathyusharns9192. You should be able to acquire morphometric measurements if you know the scaling for your images, otherwise all measurements you get will be in pixels.

Do you know how to extract the average thickness with standard deviation? Much appreciate!

@@ZiwenHe-w5s If you export your measurements into excel, you should be able to compute for the mean and standard deviation.

Hi @claireseelingbranscomb2781. Go to 3D OC Options first. Check the Maps' parameters and make sure Show numbers and White numbers are checked. To merge images, you have to convert one of them to match the other. For example, if the 3D Objects Map is 16-bit, check that your original image has the same bit-depth. othrwise, go to Image-Type and choose 16-bit.

@@Asianbabyblue17 Send me an email so I can send it to you.

@@MrUnis Macros are not my strong suit, unfortunately. Did you use the macro recorder while using the plugin?

@@MrUnis hi ! I think it has something to do with the way you installed the plugin. Are you using Windows or Mac?

@@johanna.m.dela-cruz windows

probably yes, because in 3D Treshold optimimize .....I did not get radius and outlier remover

@@nickdenniston4271 hello. Did you try the Optimize Threshold command first to determine the right settings for your image? Default settings work most of the time, but not everytime. You could also try deconvolving your images first before using the plugin.

@ ive deconvolved the images and i have also tried a variety of threshold setting indicated by the optimize threshold command. The best setting i found is a block size of 1um and a C value of 16. With these settings i am not getting as much noise and it minimizes the issue of having multiple mito being grouped into a single ROI. I am now worried that this C value is too high and i am missing information. Any thoughts?

@ I would think that a high C value is actually ideal for a deconvolved image.

Hi @BCBNarjis. How big are your images? What type are they (2D, 3D, 4D)? Are you using Windows or do you have a Mac?

@johanna.m.dela-cruz the 3D image is 108mb, and the 2D image is 4 mb. I tried both 3D and 2D threshold but they both crash the app without any prompt. I am on Windows.

Hey. I had replied to the comment but somehow it didn't post and I didn't realise. Thanks for the quick response. I figured out that it was a memory issue. I allocated more memory and now it works fine. 😊

I had a 3D image, 108mb in size and I'm on Windows

Hi @putusixtinadewandari4043. Do you mean your image is made up of several circular objects? Have you tried thresholding?

@johanna.m.dela-cruz not yet, how to do it? can you explain, please🙏

Hi @lokiiiii3333. Do you mean put your images together as a montage or merge all these images together? Segmentation is just a way to make object identification and analysis easier.

@@johanna.m.dela-cruz thanks for the reply! what i meant was to merge 5 diff. images into one.

@ You should first convert your RGB images to 8-bit (Image - Type - 8 bit). Then, select Image - Color - Merge. The colors can be modified via LUTs.

@@VaishnaviK-j5j Thanks for watching. In order to merge 2 different stacks, they have to have the same bit depth. You can change one of them to match with the other stack. Go to Image - Type - to do this.

@@johanna.m.dela-cruz Got it! It works now. Thanks so much:) Another question I had was related to why I'm unable to view the numbers on the 3D projection despite selecting "show numbers" on the maps parameters. Another strange thing is that when I view the results including volume, centroid volume etc I'm unable to see what serial numbers they correspond to. Sorry to bombard you with all these questions haha:')

@@VaishnaviK-j5j Did you check Statistics on the 'Results tables to show' in the OC Options? You can Redirect your results to your original image stack as well, just in case you are meauring intensities. As for the numbers, they are probably there...they're either too small or in a color that is similar to your object colors. Use a different LUT for your objects (e.g. glasbey on dark), The numbers are also in different slices of your z stack.

Hi @simoneceron1915. Perhaps the experts in the image.sc forum might be able to help with this: forum.image.sc/.

Hello @evanh1132. Thanks for watching. MorphoLibJ also has analysis tools. You can check out some of these tools here: th-cam.com/video/YxCacx917tg/w-d-xo.htmlsi=Rh3OIxa8B4M-f8Pf

Hi @sanyamjain4210. Are your files large? If they are not really whole slide images, perhaps you can open your files in Fiji and use StarDist to segment the cells. Also, is the pixel size really that large? Perhaps there might be something wrong with the spatial calibration in your image.

Glad this helped, @SoleneVacher. Thanks for watching.

Hi @acooper6521. DiAna is more of a 3D image analysis tool (object-based co-localization and distance analysis). There might be a different plugin that caters better to what you want to measure in time lapse images.

Hello @spanishflea634. I have no experience with SFDI, so I can't give you a definite answer to your questions. Is it considered an optical image? If it is not, then I don't think deconvolution has any scientific validation for these types of images. Perhaps what you need is an enhancement of your image (although I can't be sure about this since I don't really know what your image looks like). If you could share your image, maybe I can suggest some methods to enhance its quality.

Hi @shruthib8176. I don't think TrackMate has analyzers that measure angular velocity. It does have features that track motility type and mean directional change.

@@johanna.m.dela-cruz Thank you very much for getting back to me. I used the xy coordinates from the TrackMate to calculate the angular velocity.

@@annisadwilestari4470 thanks for supporting my channel. I could try to do a tutorial like you suggested…I just need an image to work with.

Thank you so much for answering, it means a lot, because I'm currently struggle to learn how to do it. I'll be waiting for it.​@@johanna.m.dela-cruz

@annisadwilestari4470 do you perhaps have an image i can use? You can email it to me if you’re ok with me using your image.

@@johanna.m.dela-cruz hey I'm actually have not a picture from my self. Is it okay if I took it from Journal/someone research and send you?

@@annisadwilestari4470 I guess i just need to know what exactly I’m working with or how the image would look like (since this is not a field I’m used to).

Thank you Olivia (@oliviapietrobon5225) for your continued support. What type of measurements do you want to do on your IHC images?

@@johanna.m.dela-cruz Hello, I am analyzing images where I used immunohistochemistry to mark androgen receptors. Although it's not the same, when I saw your analysis in immunofluorescence, I noticed that the analysis is done for each ROI (Region of Interest) that you create. ¿Can I do the same in the case of immunohistochemistry? Another question, when you analyze each ROI, do you get a value for each one,To create the graph, ¿did you take an average of all those values? I hope my questions are clear. Thanks, Johanna!

@oliviapietrobon5225 As long as you arr able to segment the regions you want to measure, then, you can get measurements from each ROI. I am not sure what stain you used, but if you have DAB, quantifying intensity won’t be a sensible way to characterize antigen expression.

@@johanna.m.dela-cruz Hello, I use de tunel technique and caspasa 3 (for this i use DAB), now another question, I see that you're making a graph of the measurements; I'd like to know if you're calculating an average of the values from each ROI. My concern is that sometimes the values can be very different between each ROI, wouldn't that result in a high deviation?

@@oliviapietrobon5225 yes, it's an avaerage.

Hello @Lam-m3l. Thanks for watching my tutorial. Unfortunately, due to some permission issues, I can't share the images I used. However, there are several image datasets that you can use from this site: imagej.net/plugins/samples.

@@johanna.m.dela-cruz Thanks for the info. I was interested in the convoluted-looking fiber-like first dataset that you used. Not necessarily microglia though.

@Lam-m3l You’re right…that was a z-stack of microglia.