How to plot profile intensity for multiple fluorescence images in ImageJ

[@AliceritaE]

Update 18/08/2022: To generate the profile plot, go to PLUGINS >> GRAPHICS >> RGB PROFILE PLOT. It seemed the path was modified, you can then proceed as shown in this video was You can download the RGB profiler here; imagej.nih.gov/ij/plugins/rgb-profiler.html

[@melissatraver7539]

Hi Alicerita, thanks for the great video! When I use the plugin, I only see Plugins > RGB Profiler (no graphics tab) and it seems like the output is a TIFF file that's not possible to edit. Am I missing something in my process, or has the plugin functionality/output changed?

[@AliceritaE]

Hi Melissa, thank you for your comment. Indeed, the RGB profiler seems to be broken. Please go to Plugins >> Graphics >> RGB profile plot. Then continue to edit the profile plot as shown in the video.

[@melissatraver7539]

@@AliceritaE thanks for the reply! After I installed the plugin, it does not have the plugins >> graphics path, so it may be that the installer is also broken?

[@AliceritaE]

Did you restart the imageJ after installing the plugin. A new plugin might be needed for graphics. Let me browse and get back to you

[@96sharmistha]

@@AliceritaE I am also having the same problem. What is the solution?

[@vinalestariputra6770]

How amazing! Thank you so much Alice, for your effort in putting this together and sharing with other fellow researchers!

[@AliceritaE]

Glad it was helpful! cheers!

[@Nieukoop]

Hi Alice. Thank you so much for this very informative tutorial. I could not find the Graphics plugin in my list! I dowloaded the RGB profiler, which is. working, but. I cannot make changes in the graphics. could you please provide the link for the Graphics plugin for download. Many thanks in advance

[@AliceritaE]

Hi, here is a video on how do that without the RGB plugin. kzbin.info/www/bejne/i4fVlayhhLSSatE

[@kehindemedase5242]

Highly informative and educative! 🙌👏🌿

[@AliceritaE]

😊 thanks

[@illyada]

These videos are great and so clear!

[@AliceritaE]

Thank you @illyada, I'm glad you found it useful

[@artyorange7]

thank you for the tutorial! it was super useful! :)

[@AliceritaE]

You're welcome 😊

[@jordanquellec6878]

Hi, thanks you so much ! Really helpfull ! You made it so easy ! Thanks again !

[@AliceritaE]

Hi Jordan, I am delighted that you found the tutorial useful. Cheers!

[@abdouliejallow1227]

very informative. I am trying to use the method to quantify staining in immunochemistry (gold nanoparticles conjugated with antibodies). so in this plots, what is the quantity of the grey value? and what is the background? how do i substract the background from the grey values?

[@AliceritaE]

After drawing your ROI on the sample, go to Analyze (select measurement type) the click on the measure particle option

[@AliceritaE]

Hi @Abdoulie jallow, were you able to do the analysis

[@abdouliejallow1227]

@@AliceritaE hello Alicerita, thank you very much, yes i did some of it! The good news is that it is working great!

[@AliceritaE]

I'm happy to know that it's working great. Have a lovely weekend🌻

[@raziasultana8103]

@Alicerita. Thank you for the detailed information. Do you have any tutorials for radial profile analysis in Fiji. Please provide a tutorial for intensity analysis using the radial profile plugin.

[@AliceritaE]

Hi Razia, You're welcome. I don't have a tutorial on radial profile analysis. I can however check it out and record one. Thanks for your kind feedback.

[@raziasultana8103]

@@AliceritaE Thank you so much!!

[@AliceritaE]

Hi @Razia, I'm sorry for the delay, I have been busy with my thesis. I will prepare the radial tutorial and upload by the weekend. Thanks for your patience

[@georgey-b6080]

​@@AliceritaEIs the radial plot available?

[@dinelkamadhushani460]

Dear Alicerita , thank you so much this is very useful for me. Also I need to analyze a video. What I need to do is I need to plot the light intensity of images with time in fluorescence video. Could you please let me know can I use this image J to do that?

[@AliceritaE]

Hi Dinelka, You can first split the video into frames. then quantify each frame to get the intensity. you can then put the values foe each frame (time) together in excel sheet and then plot over time. i.e Y axis = intensity X axis = time (frame)

[@dinelkamadhushani460]

Hi Alicerita, Thank you for your reply .It is more informative. I'm a student and new to imagej. I got some problems when import the video into image j. My original video is .mov format. But that format is not supported to imagej. So I rename it as .avi format and tried to import. Then I got an error message " An error occurred reading the AVI file". But when I try to import a segmented video in .avi format it was successfully imported. But I need to analyze the original video. Could you please let me know is there a specific way to import my video into imagej?

[@AliceritaE]

Dear Dinelka, I would suggest to install the reader plugin and then try to open your video again. Here is a tutorial in how to install the reader. kzbin.info/www/bejne/moDOpqRjrs6YrM0

[@dinelkamadhushani460]

Hi Alicerita , I did it. Then It didn't show any error message. But the video didn't open.

[@AliceritaE]

Hi Dinelka, I am glad you are not receiving the error message anymore. Do you by chance have the original .mov file before you renamed it to .avi The renaming might have changed the compression format and that' might bewhy imageJ is acting up. Could you please try the original video. If the problem persist, you might need to restart your computer. Cheers

[@ikenganonso6789]

Really helpful. Thanks a lot.

[@AliceritaE]

You're welcome.

[@mahmutmijit7316]

this is very helpful. i would like to learn how to do a profile plot of multiple-lane of western blot membrane. do you have any tutorial? or could you please give me any instructions? thanks!

[@AliceritaE]

Thank you for kind comment. You can use the rectangular shape to draw around the the band you want to quantify, press T to add the ROI to the manager. Do this for all the bands. Next select the first band, go to analyse >> Gels >> select first lane. Then select the second ROI on the next lane, go to analyse >> select next lane (and repeat the whole process again until you have selected the whole ROI) After, go to analyse >> plot lanes (this will give you the peak of all the lanes)

[@AliceritaE]

Hi @Mahmut mijit, have you been able to do your quantification

[@sultansahinkeskin8500]

Hi Alicerita, Thank ypu so much for this very useful tutorial. I am curious about how I can calculate sum of gray value in 2d plot profile? Thank you so much

[@AliceritaE]

Hello Sultan, you are welcome. You can export the the plot in .CSV to calculate the sum. But my preferred way would be for you to draw the region of interest. Then go to analyse >> Set measurement//tick sum of grey value and mean grey value. Then go to Analyze >> measure

[@hebaali4446]

great video thank you!

[@AliceritaE]

Thanks for your kind feedback

[@amulyaramakrishna2803]

Ma'am, thank you very much for the tutorial. I am unable to find 'graphics' option in the plugins. Could you please let me know how to access this option? Thank you

[@AliceritaE]

Hello Amulya, I have experienced that before, you can download the graphics plugin or use another method described I'm this video here, let me search for the link

[@AliceritaE]

kzbin.info/www/bejne/onunhniPgrt4obs

[@amulyaramakrishna2803]

@@AliceritaE Thank you, ma'am. Could you let me know how to download the graphics plugin as I was unable to find it in the video? I wanted RGB plot profile, so I downloaded the RGB profiler directly from the website without downloading the Graphics plugin. But, in RGB profiler I am unable to see the options such as 'more', 'data' etc. So could you please help me with this?

[@AliceritaE]

Please give me some minutes to check

[@AliceritaE]

@@amulyaramakrishna2803 Hi, Here is the link to download the RBG imagej.nih.gov/ij/plugins/rgb-profiler.html After dowloading the plugin, drag and drop it to imageJ so that the software updates. after that, close and restart the software. then go to PLUGINS >> GRAPHICS >> RGB PROFILE PLOT.

[@mauromontalbano6300]

Hi Alicerita, very informative and well-done tutorial, what if the channels are four or more? Thank you

[@AliceritaE]

Hi Mauro, If you have more than three channels, please watch this video kzbin.info/www/bejne/onunhniPgrt4obs

[@BushCampingTools]

Hi great just came across your channel. Can I make a profile plot for say 100 consecutive images and get image J to do them all at once using the same ROI and of course in the same position as i want to measure intensity change over time? I know I can simply put them into say Adobe AE but and get a line plot but with that software it is not appropriate for any sort of quantitation. Or can I process a batch if images and take the average intensity (or one obtained by using a ROI) as I want to look at intensity changes over time. Sorry for the dumb questions. I'm a confocal guy. I subbed too BTW.

[@AliceritaE]

Hello 👋 Thanks for your kind comment. I will like to know if the image is a time-lapse. If it is, it should be possible to measure the ROI over time and played into an animation. Alternatively, you can use the average intensity overtime

[@muskangupta4542]

Hi Alicerta! Thank you for the informative vedio. Currently, I am doing Co-localization for my images. I downloaded the plugin RGB profiler. However, it does show the option in the end like 'data' or 'save'. How do I save my data in excel sheet?

[@AliceritaE]

Do you mean it doesn't show the option?

[@AliceritaE]

I'll check it out and make a new video

[@AliceritaE]

Expect a new video today. I have checked and confirmed that the old method is ko longer working.

[@muskangupta4542]

​@@AliceritaEMany Thanks Alice! Looking forward to the video 😊

[@AliceritaE]

@muskangupta4542 it's online now! Thanks for watching

[@paulina369]

Hi, thank you very much for your video. I have been using the confocal microscope, measuring DAPI and GFP. I have so far measured the intensity if the whole image (like in your video), however I need to recognize each GFP/DAPI spot and compare. Also I have managed to create a ROI with my selected spots of DAPI (and use the same ROI for the GFP) and I was trying to use Analyze-plot profile like in your video to get the intensity, however this do not work since it needs rectangular or linear settings. How can I proceed with this? Thank you!

[@AliceritaE]

Hi Paulina, do you mean that you want to plot profile plot using a rectangular ROI instead of the line tool? I haven't done that before, I have only quantify signal within a box or rectangle and then plotted the intensity using bar chart.

[@paulina369]

@@AliceritaE Hi! No, I wanted to quantify every single dot. But I needed to create a selection of the dot and add it to the manager, so that I can use the picture 1 to compare Picture 2 on the same positions. It seems I solved my problem, I had to split the channels, then I adjusted the threshold using Otsu, followed by creating a Selection, then I can quantify normally using the gray scale value. Thank you so much for your videos though, they are so well explained and easy to follow. Best, Paulina

[@oluwatomisinfaniyan2715]

@Alicerta. Thank you. The video is highly resourceful. However, please do you have the link to download the "Graphics plugin" without downloading a new FIJI software

[@AliceritaE]

Hi Oluwatomisin, Do you mean plugins for graph? imagej.nih.gov/ij/plugins/graph/index.html

[@oluwatomisinfaniyan2715]

@@AliceritaE thanks...yes

[@AliceritaE]

You're welcome

[@oluwatomisinfaniyan2715]

@Alicerita, thank you for the plugin but the plugin did not display the plot profile you explained in the video Also, please why did you draw a line across the image at the start of the tutorial Thanks for your assistance

[@AliceritaE]

The line I drew is needed to measure the intensity across that line. I can check Fiji when i get home. I know that the paths are different for Fiji compared to imageJ

[@sweetymeel4630]

hey thanks for the video! i am unable to get rgb profile plot. rgb profiler doesn't provide tweaking the axis scale etc options. any idea how to get it?

[@AliceritaE]

Hello, I will check it out at my side. In the mean time, you can check this other video. kzbin.info/www/bejne/onunhniPgrt4obs

[@AliceritaE]

You can download the RGB profiler here; imagej.nih.gov/ij/plugins/rgb-profiler.html

[@shiyingliu2174]

I installed the RGB profiler from the above link, however my plugin does not show the "more" option to allow me to change the content style:( where are they?

[@AliceritaE]

I will check in 30min and let you know

[@AliceritaE]

Hi Shiying Liu, I just checked it now. You are right, the option for editing it is gone. I will update my imageJ to see if there will changes

[@AliceritaE]

@@shiyingliu2174 I have now updated this. Go to PLUGINS >> GRAPHICS >> RGB PROFILE PLOT.

[@um7144]

in X axis (distance), How to determine as - coordinator (unit: micron, um)

[@AliceritaE]

If you are working with the original file data from the microscope, it should contain the meta data. Thereby automatically calculate the number of pixels that makes a micron unit.

[@azeezishola2912]

Thanks for this lecture but how about fluorescent images of 4 colors? I have confocal images of 4 channels

[@AliceritaE]

I have never profile 4 channels. I will write you of I find something. Cheers

[@azeezishola2912]

@@AliceritaE thanks

[@AliceritaE]

I was wondering if it's possible for you to split the image into 2 channels each

[@AliceritaE]

Meaning Red and green and then blue and yellow. That way, you can flash colour the last channel and then use the RGB profile plot. You can send an example into my email. Then I will record my screen and send it to you. That might be the easiest way to do a profile plot for 4 channels

[@AliceritaE]

Hi @Ishola, For the 4 channels, measure each channel separately using the same ROI. that way, you can combine the result of the four channels in excel to plot the graph.

[@marianaholguin314]

Thank you so much!

[@AliceritaE]

It's a pleasure, thank you for watching

[@ayelentayagui6658]

Hi Alicerita, thanks so much for this tutorial it has been very helpful for me. However, I need your help or anyone else, as I can not get the graphics plugins on my Imagej version. I tried installing a new version, it has the graphics plugins but I lost many others plugins that I really use and need for my research, so if you could please guide me how to install, just that plugin to follow your video, it will be awesome. Thanks

[@ayelentayagui6658]

Never mind, I sorted out myself...I encounter another problem now...When I try to do a composite of my red and yellow colors, it doesn't show me those colors, it appears like bleached, I have not idea what is going on, I have never got any problems in the past when I did composite, splitting or merging channels, but now, all my composites look bleached or only show me one color. If you can help me with this, it will be great. Thanks.

[@AliceritaE]

That's great, I was just going to use my computer to do the troubleshooting

[@AliceritaE]

Hi, did you select yhr right colour for each channel

[@AliceritaE]

Are you using ImageJ or Fiji

[@AliceritaE]

Can you pls check if your imageJ is up to date to the latest version. It's unusual to get bleached images

[@MoinKhan19]

After creating the composite image, I am getting an error that image must be RGB for the plugin (RGB Profiler) to work. But in your video, I can see that RGB Profiler is working fine with 8-bit composite image. Could you please explain this discrepancy that I'm facing.

[@AliceritaE]

HI Moin, After making a composite, go to image // Adujst// RGB color That will generate a RGB image for you. Alternatively, When merging the channels, you can unchecked the composite option and click okay.

[@MoinKhan19]

@@AliceritaE Thanks a lot for your quick response. But I'm not getting satisfactory plot profile with RGB image. The peaks are getting saturated at 255 and look flat at the top. Is there any other plugin to plot profile intensity for multiple fluorescence images?

[@AliceritaE]

Yes, RGB profile plugin

[@MoinKhan19]

@@AliceritaE RGB profile and RGB profiler are two different plugins?

[@AliceritaE]

They are the same

[@rosella5589]

Hi Alicerita, This is so helpful! Thanks. Can I ask you how we can change the x-axis value from inches to pixels in RGB profile plot gotten from composite image? My RGB profile plot shows always as inches in x-axis. I couldn't find yet a way to change it.

[@AliceritaE]

Hi Rosella, You can first calibrate your image to microns or pixels. The reason my plot was shown in pixel was because the image was in pixel. If you set the scale to microns or pixel, then your plot will automatically pix the same format. Cheers!

[@rosella5589]

@@AliceritaE oh Thanks Alicerita~~!!^^*

[@AliceritaE]

You are much welcome @Rosella

[@etelgimba1]

Dear Alicerita, could you please provide the steps to install the "Gaphics" plugin, i tried tio update to Fiji, but I could not achiev istalling it, thanks ! I am using on a Mac

[@AliceritaE]

Hi, by default, Fiji should already have that. I will record a video and upload it today. Cheers!

[@AliceritaE]

I have recorded the video. It shoiuld be online in 10 mins

[@AliceritaE]

kzbin.info/www/bejne/Y2bFpISlq7edsNU

[@etelgimba1]

Dear Alicerita , thanks a lot for Kindly recording this vídeo , tomorrow morning I Will test. I love your videos , congratulations !

[@etelgimba1]

Dear Alicerita, I folowed all the steps you presented and tried it , but unfortunaly another message appreared and I also could not install the app. The message after Ïnstal" was someting like: "kava.io.Filenot FoundException

[@AkhabueKennethOkojie]

So, what is the biological relevance of this graphical plot?

[@AliceritaE]

A profile plot is usually used when a researcher wants to know the proximity of two closely localized proteins or reporters. Also, it can be used to show the expression level of a particular protein, especially when comparing the expression of a wild-type versus a genetically modified strain.

[@AkhabueKennethOkojie]

@AliceritaE Thanks for the information.

[@johnsmalley8853]

Can this routine be done for 6 images ?

[@AliceritaE]

Yes! I have another tutorial where I showed how to do it for 5 images.

[@AliceritaE]

Hi John, Here is the link for more than 4 images kzbin.info/www/bejne/onunhniPgrt4obs

[@johnsmalley8853]

Many thanks Alice. The videos are so informative. Much appreciated! Can you do a similar routine to examine a single point of interest (a single pixel ?).

[@AliceritaE]

Yes, you can routinely check a single point of interest. All you need to do is to add that region to a Region of interest ROI manager. Save the ROI manager and routinely open it to load unto a new image

[@koungngeun8055]

Hi Alicerita, Thank for your wonderful sharing. Could I ask you how to check the red stain intensity of the cell in the image and count the cell number? The stained cells are differentiated cell and so I would like to know the average differentiated stain cell and unstained cells number. Sorry, I could not comment the photo. I will send you in your FB chat box. Please check it for me.

[@AliceritaE]

Hi Koung, I acknowledge receiving the images in my Facebook messenger. I will experiment with them and get back to you. cheers

[@koungngeun8055]

@@AliceritaE Thank you for your kindly reply

[@wenjingwu6293]

What plugin are you using?

[@AliceritaE]

Hi Wenjing Wu, I am using Graphics plugin.

[@AliceritaE]

you can download it from here imagej.net/plugins/rgb-profiler