Hello thanks for reaching out. Please use %area for the quantification

Hello thanks for reaching out. Please send me an email at [email protected]

Hello, thanks for reaching out. Yes you can find the CTCF for hoescht only stained image. image split may not be needed if your image is showing only hoescht staining. DAPI channel can be used for this imaging. but you still need to change the image into an 8 bit image

Hello, thanks for reaching out. A large size >10MB image may cause this problem. please try reducing the file size under Image, adjust and size and then run the process again. Hope this helps you. Let me know if it worked. All the best.

@@nrttaye4033 ,I would like to thank you for your quick answer. And what the best extension of the figure to be read by the macro?

both jpeg and tiff are acceptable. i think TIFF resolution is better than jpeg

Hello, I am not sure how to convert %Area into Intensity. This plugin is good for unmixing dyes in images when colors combine subtractively (bright field microscopy with histology stains, watercolors, or printed material with transparent inks). This is the reason It is unsuitable for fluorescence microscopy (fluorophores mix additively) or reflectance imaging that is intensity measurement from %Area. Quantifying staining intensity is only useful when dye uptake is stoichiometric (e.g., the Feulgen reaction for DNA). Unfortunately, immunohistochemical methods are not stoichiometric, making them inappropriate for measuring antigen expression (using intensity).

Hello, thanks for reaching out. Any part of the gel area you consider as background can be selected for subtraction. The idea is to get clear away the region near the dna band.

@@nrttaye4033 what happens if I don't subtract the background? is it still valid?

If the gel bands you generated is clean with no noise then background subtraction is not required. this is mainly due to three reasons 1. accuracy in quantification: Accurate quantification is based on band intensity measurements. Background fluorescence or staining can falsely exaggerate these values, resulting in erroneous quantification. Subtracting the background guarantees that just the genuine signal from each band is assessed. 2. Consistency in the dna gel band: Background levels can differ between images due to illumination, exposure, or gel staining. Standardizing background subtraction yields more consistent findings across multiple studies. 3. if you have to compare between several gels: Consistent background subtraction provides for more trustworthy comparisons by reducing variability in background levels.

Hello, thanks for reaching out. could you please delete the plugin from the plugin folder and then re-install it? I have used this plugin only in ImageJ and have not tried using Fiji. Sometimes updating ImageJ to the latest version could also be an issue (plugin compatibilty). If this is the situation in your case, reverting to older version of ImageJ helps at times.

Hello, thanks for reaching out. The measurement is based on selections. Selections can be done either using the Wand tool like you mentioned as well as using the command options as demonstrated in this video. Either way is correct.

​@nrttaye4033 Thanks! I am facing problem in selecting more than one cell using the wand tool. Can you help how to troubleshoot that?

Hello. Please use this formula to convert pixels to mm. millimeters = (pixels X 25.4) / dpi. Now the millimeter can be converted to micrometer. You may also find calculators online to calculate it automatically.

Hi here is another video that might be of interest to you m.kzbin.info/www/bejne/javSipVvird6oZY

Hello, please have a look at this video kzbin.info/www/bejne/mZSQk6WvoMScntk

Hello, the link does not work anymore. Please send me an email at [email protected] and i will send you the plugin

Hello I know that the output is either in %Area or Relative density. ImageJ uses the percentage area for Western blot quantification to calculate the percentage of total area for each band in a row. This is useful when there are numerous bands per lane of various proteins and cannot be estimated as an integrated density.

Hello, thanks for watching my videos. Use the selection tool of your interest eg, the rectangular selection tool and select the region of interest. Now run the plugin as shown in the video. All the best with your analysis.

Hello thanks for watching my videos. I just tried with a stack image and it is successful. The steps are same for either for a single image or stack images. Let me know if it works at your end.

Welcome! Here is another video to automatically convert in a batch mode kzbin.info/www/bejne/mZSQk6WvoMScntk

You are welcome. Glad you found it useful

Hello thanks for reaching out. Here is the original publication for the angiogenesis analyser imagej (Angiogenesis Analyzer for ImageJ - A comparative morphometric analysis of “Endothelial Tube Formation Assay” and “Fibrin Bead Assay”) The link is www.nature.com/articles/s41598-020-67289-8 . Let me know if you have more questions.

@nrttaye4033 Thanks for the paper, but it doesn't say tho like what the stats signify .and what is the most important factor among them to look for. If you can explain on that stuff it would be great.

Hello, you are welcome. I am not sure about that. Could you please send me a sample image at [email protected] I could troubleshoot it

Hello, this works only for Fiji and not ImageJ. Please use the latest version of Fiji and then try again. Let me know if this still does not work.

Hello, i guess the link does not work anymore. please send your email to [email protected] and i will send you the plugin

Hi the link does not work, could you please send me your email [email protected] i can send you the plugin

You are welcome. Glad you found it useful.

You are welcome. Glad you found it useful

Hello, thanks for watching my videos. I appreciate your insightful observation. You brought up some excellent points regarding if 3D image generated using ImageJ is merely a representation of the 2D image. It is true that some morphological aspects and substructures are highlighted in the 2D image, which can be important for understanding what is being displayed. I think with this 3D representation it may provide us with the ability to see the depth that increses the understanding of the spatial arrangement and interactions among structures. 3D rendering may offer insights into the morphology of complicated components by exposing internal features and layers (users can rotate the 3D images) that may be hidden in 2D. Let me know if this answers your question.

hello, you are welcome. please try changing the parameters in the wound healing tool options especially the "min size" and "radius close". The parameters in the video is used with the unit inches. if you have unit of images in pixels, then need to change accordingly. let me know if that worked, esle you could send me a sample image to troubleshoot.

@@nrttaye4033 Hi thanks for your quick response. the unit of images are in pixels in my case. Would it be possible to change them into inches? I tried to change the "min size" and radius close but not successful in analyzing the data.

@@nrttaye4033 Also, is there any option to upload the sample picture for you?

please mail it to... and i will try to troubleshoot it

@@nrttaye4033 image sent. Thanks so much for help!

Hello, as far as i know this macro does not work for z stack.

Hello, please provide you email. I can send you the plugin

Hello, i guess the link does not work anymore. please send your email to [email protected] and i will send you the plugin

This methods is applicable for analysis that involves "limit to threshold" option selection in the "set measurement window".

Glad you found it helpful. Thanks for watching and subscribing

Hello, welcome. it is easier to convert into mm using the formula: mm = ( pixels X 25.4 ) / DPI. you can find this formula online

no it will not work. I will write a macro and post it soon

Hello thanks for the enquiry and the comments. I regret to let you know that I do not offer one to one tutorials, however i can help you over emails.

Hi, here is your solution. Select the region of interest using any of the selection tools in the image and then click on edit and clear. have a look at this video from 4:22 to 5:20 mins kzbin.info/www/bejne/e3vEl4CqiteciNk

Hello, I tried doing a z stack and it was not successful. another way to do is to first convert the z stack images into individual images and do a batch process.

I want to measure length, width and straightness of actin fimaments. I need some suggestions

Hello, JFilament is an option. I do not have videos on this. please check www.lehigh.edu/~div206/jfilament/index.html for more details. If your filament is short or curve and need to mesure length or morphology i have some videos on this kzbin.info/www/bejne/eYGVcmCAqZdqb8U kzbin.info/www/bejne/mGS9Y6dsZ85setU

That would be from the scale bar of your image.

@@nrttaye4033 In this case, would it be possible to convert pixels to mm? For example, 312 pixels = 82.55 mm? Thank you for your videos, they are very useful for analyzing my research data

Hello, this formula is used for conversion mm = ( pixels X 25.4 ) / DPI. You can find this formula online

Hello, thanks for reaching out. Try changing the threshold value ranging from 50-150 in the wound healing tool options window and then run the macro again. The threshold value depends on the contrast of the cells and its background. sometimes the image dimensions are in pixels or inches and radius close value needs to be changed accordingly. If still it does not work out, send me a sample image to troubleshoot it.

@@nrttaye4033 Hi What's your email ID to send a sample image? I am still not getting

Hi thanks for reaching out. it did not happen to me so far. Can you send me a sample image? I will try to troubleshoot it.

@@nrttaye4033 Hiiii, thanks for answering me :D I was messing around with Fiji and ended up finding the setting described below: image> adjust> auto crop (guess background color) I put it in macro and it worked, thank goodness. anyway, thanks for the guidance, you always help me. all the best!

You figured it out, Excellent! All the best with your analysis.

yes it works. The ROI manager will pop up after running the macro. Using the ROI manager window you can identify which nuclei are being referred to.

Hi, thanks for reaching out. Here is the link to download ImageJ. choose the version that suits your operating system imagej.net/ij/download.html

have a look at this video from 0:00 to 0:45 seconds, it shows how to install imagej

hello, thanks for reaching out. Does the graft look like hairs with certain length or just transplanted with round shape structures? I may need to know how the transplant images looks like before coming to a conclusion. If they appear like cilia/filament i have video tutorials on how to count these kzbin.info/www/bejne/mGS9Y6dsZ85setU if you have overlaps then try "ridge detection" plugin github.com/thorstenwagner/ij-ridgedetection let me know if these helps. Sure happy to help.

@@nrttaye4033 hello again , can I send for you one of my plate with specific number of hair graft ?

Can I have your Email address?(for send a image of graft)

here is my email

@@nrttaye4033 thank you .I sent for you an email

you are welcome. listing intensity of each cells would require a ROI/specific boundary/selection of each cells while thresholding. in this video the selection command uses "select all". However, to do individual selection either the cells do not adhere/touch one another or a manual seperation would be needed. For instance the DAPI is already seperated in this video. during the analyze and measure steps check the "add to manager" option in the ROI manager window to get individual cell intensity. Here is a demo video kzbin.info/www/bejne/nqHbn5WAlJ14e5Y where i analyzed the intensity of each DAPI seperately.

The plot results instructs the Weka core to generate the model performance chart, which includes curves for ROC, precision/recall, and other metrics based on the training dataset. These curves demonstrate the classifier's performance based on the many thresholds that can be applied to the probability maps. A receiver operating characteristic curve, or ROC curve, is a graphical figure that shows the performance of a binary classifier model (which can also be used for multi-class classification) at different threshold values. The ROC curve plots the true positive rate (TPR) vs the false positive rate (FPR) at each threshold setting. The ROC can alternatively be viewed as a plot of statistical power vs Type I Error of the decision rule. The ROC curve represents the sensitivity or recall as a function of the false positive rate. Precision and recall are performance measurements used in pattern recognition, information retrieval, object detection, and classification (machine learning) to assess data recovered from a collection, corpus, or sample space.Precision (also known as positive predictive value) is the proportion of relevant instances among the recovered ones. Recall (also known as sensitivity) is the proportion of relevant instances that were recovered. Relevance thus serves as the foundation for both precision and recall.

The previous video showed how to convert the czi files into individual jpeg images. Using this method is laborious and time-consuming. The macro code in this video is quite useful for batch processing. I hope this macro is saving many users.

Hello, I think you can set scales. Have a look at this publication onlinelibrary.wiley.com/doi/10.1111/srt.12634 let me know if this helps.