Counting Cells with ImageJ
HTML-код
- Опубликовано: 9 сен 2013
- Keep in mind that ImageJ processing is not perfect. However, the error of missing cells may be "corrected" by the error of including some noise.
Steps in Video:
Process - Subtract Background
Image - Adjust - Threshold
Process - Binary - Fill Holes
Process - Binary - Convert to Mask
Process - Binary - Watershed
Analyze - Analyze Particles
Basic description of tools:
Subtract Background - cancel out noise of the background
Threshold - change the image to binary image of red, black and white, or blue
Fill Holes - fill empty spaces between rings to make circles
Convert to Mask - allows for subsequent processing
Watershed - automated separation of separate fused cells by a 1 pixel line
Analyze Particles - process the image to acquire a cell count
Size - parameter of what cells to include in data by area (pixels^2)
Circularity - parameter of what cells to include by how close to a circle the shape appears. The maximum bound (1.0) means a perfect circle. The minimum bound deviates from being a circle.
Show - Outlines will trace the outside of the mask images. Masks will show the black and white image similar during thresholding.
Exclude Edges - ImageJ will not include cells that are not fully contained in the boundaries of the image
Macro Code:
run("Subtract Background...", "rolling=12");
setAutoThreshold("Default dark");
setThreshold(35, 255);
run("Convert to Mask");
run("Fill Holes");
run("Convert to Mask");
run("Watershed");
run("Analyze Particles...", "size=120-Infinity circularity=0.00-1.00 show=Outlines display exclude clear summarize");
Thank you for explaining how to process an image through ImageJ and uploading a video about it!
and I believe you need to revisit your video whenever there are new comments to answer them...again thanks!
Outstanding Tutorial! It just solved my problem. Thanks a lot, Kevin. :)
Thanks Kevin. It made my job a lot easier.
Thanks! Trying out image J for the first time.
thanks this makes measurements so much easier
Thank you very much for that quick tutorial that helped me a lot !
That's sooooooo useful!! Thanks A LOT!!
Thank you so much! ❤️ This really helped me a lot ☺️
Thank you! This was very helpful!
WONDERFUL!
Thanks very helpful
It was really useful. Thanks a lot!
This is what I looking for.. Thank you
Thank you very much! Very useful
Thank you very much for the great video
Thanks very much for sharing! this is very helpful!!!!
You are amazing thanks a million!!
Thanks! That's great, I'm trying for geological material!
Hello Kevin
Thanks for the video. It is very informative.
Do you think the image file must be (tif) in order to use the watershed function and subsequently you can count the objects using the analyze particle command?
Thanks so much!!
Many thanks for the video, it helps me a lot. I wonder that if I have cells stained with Hoechst, and I want to count dividing cells with 2 nuclei close to each other (at anaphase or telophase) as 1 cell, what should I do?
hi Kevin, may I ask if I can choose the area I am interested in to count the cell number? do I have to count all the cells in the figure?
Thanks for the video! It's excellent!
I have one question: I have to quantify some PI (propidium iodide) images of hippocampal slices... how do I get the percentage of dead cells for the total tissue area? For that, how do I get a sum of the area of all dead cells and divide by the total area of the slice?
Preferably in a quick way without having to mark all dead cells.
Thank you!
Thanks for the video!
In the results table - how did you manage to get the Perim., Circ., AR columns? I have just Area, Mean, Min, Max. (also I don't see the center of a cell - just the borders). Thanks for any help
On Results window press the button "Results" and then choose "set measurements".
It's been 5 years since your question but maybe I will help someone else.
Does this work for 3D images as well? As in if i have several slices of pictures?
Thank you for this!
Hi there. I'm looking for a program to count cells, but this program doesn't seem to do the trick. I have images of a micro-array covered with wells and I only want to count cells _inside_ those wells (which are regularly interspaced as if located on the vertices of a grid). In other words, I need the program to only count cells on particular pre-selected locations of the picture. Any advice there?
good!! thanks
Thank you for the informative video. Is there any way to batch this if we have many samples to count?
many thxs for the share
You saved my master degree
Nice video.
Hi! Thanks for the video. When I put the watershed part my software is just binding black points... Its not working as the video said.. can you help me?
Hi. Thanks for the video.
How can I use imagej to count islets of langerhan?
What exactly does the image show?What kind of cells?I am writing my thesis and it could be very helpful to have more information about the picture.Thanks a lot in advance.
Nice tutorial video, it's very informative. I have a question if you don't mind. Is there a functionality or option in ImageJ that allows the user to count the number of pixels of a particular color and/or get its percentage from the total number of pixels?
For example, I have an image with color A and B, I want to get the total number of pixels of color A, or color B. (For simplicity, we can let color A = black, and color B = white)
I would really appreciate if you could respond to my queries, it will be a great help. Thanks in advance.
You can paint B color as red in histogram menu and then count the area of red color in Results table.
Gracias!!
Hi Kevin, good job, I was wondering if it is possible to count cells in more than one image at once.
for example, I have 200 images and I want to cell counts from each image at once.
Thank you in advance!
were you able to figure it out? I have the same question
@@alexandralramosfigueroa4501 were either of you able to figure it out ? I also have that question lol help
were you able to figure it out? i am in the same situation
Thanks
Hi, I have a question and hope to get my answer from you.
Actually, I have some pictures (with green florescent dots as cells) randomly taken from my sample. I counted the cells/picture by ImageJ and I was wondering how to extrapolate these certain number of cells counted as it's very hard to take lots of pictures from my samples by a florescent microscope.
You shouldn't really extrapolate like that because you're assuming even growth across the plate/well. If you have to, then you really should take 5 random images and take the average of those 5 images. After this, divide the total size of the well/plate by the frame size of the image then multiply this factor by your cell-count. Ie:
Image frame size = 2mm^2,
well size = 100mm^2
Average cell count across 5 images= 200
Total cell count = 200 * (100/ 2)
= 10000 cells
Kevin, How does someone acquire the photograph of a sample that is stand. I mean if I want to count cells per ml, how do I know how much sample is in the photo.
I know people say use a hemocytometer but is there a glass slide with a groove that takes a desired amount of sample or something other than a hemocytometer. I would have thought a hemocytometers lines would mess up the image taken from a microscope.
Thanks ..................
Does anyone know why images are first converted to binary images before analysing them? Why is it not possible to do it on the grayscale picture?
I am interested in counting pollen grains and also pollen types differentiated using staining dye for viability. However using the methodology as shown I am still not able to get the exact results and colour differentiation not possible. can you describe the steps if I mail you a sample pic. I shall be highly obliged.
why don't you try with MTT dude? (Dafni's method) it's nice and easy....but i also have a problem of how to count :(
Thanks alot! Fill holes doesn't work well for my particular images - the middle area of cells disappears completely and the outer cells don't change. Eh. It still is working well - I used the 'record' feature to make a macro to automate it and it works like a charm
edit, I see you included macro code - yep. any luck running this in batch?
I would like to count just pairs of cells (2 cells that are close to each other and no cells which are standing single) Do you have any clue how to do that with ImageJ?
thanks...
Nice video, I want to count spherical shape cells. But the sizes vary from each other might be small as 10um diameter to too big diameter. Can you help me to count all the cells
Thanks so much for posting this! Is this method attached to any publication that I could cite?
Thanks so much. Please I do I calibrate my sample. Please how do I record my my values
Hi. Thanks for your sharing. It's so helpful! I have a question about watershed. I can't separate cell one by one. Are there some better method to separate cells?
Did you figure this out? I am trying the same
Thank you so much for your woderful tutorial.
I have a question. How I can know the exactly threshold?
and how many pixel I should use for subtract background in each photo? Is it the same?
How does the "Fill holes" algorithm at 2:07 work?
Hi what magnification did you get your image on?
Do I buy a glass slide with a well and inject my stained sample into this well prior to taking photo.
if I want to erase something, how do I do that?
Hi. Thank you for showing this. it's very good. However this is good for counting round cells. Cells in culture with different morphology are more complicated. Does anyone have a clue how to count fibroblasts?
+Mauro Cafundó de Morais Use DAPI and count nuclei
hi, could you please give me the link of microscopic blood image..
thx for the tutorial. btw, i have an image that consisting of dirt. if dirt > 0.04 mm need to count, if below 0.04 mm not count. where i can put this parameter? thx
And how do you actually count the number of cells??
What is the size of the cells? I mean, the diameter.
nice info, but how to cite in journal?
Hey guys! Wish this message finds you well. Is there anyone here have the text tutorial of this software so he could share it with me via my e-mail please? I need it a lot to estimate the severity of bean anthracnose for my thesis. Thank you in advance!
If someone can tell me how to count stomata; that'd make my life waaaayyyy easier! ;)
Thanks very much! It's really helpful.