Green Flourescent Cells

cellprofiler

#1

I’m new to CellProfiler. So I’m not sure how to use it. I have an image of green flourescent stained cell and red flourescent stained cell. Could any one give me instruction step by step to set up in CellProfiler to cound green flourescent cell and red flourescent cells? Thank you so much for you help.


#2

Hello,
Would you mind giving me a bit more background on the green and red fluorescence (what is stained green and what is stained red)? Are you trying to quantify a ratio of green to red? Are your images false-colored or grayscale?

thanks,
martha


#3

The green flourescent cells are caspase 3 expressed cell. The red flourescent cells are necrotic cell stain with PI. I want to count the ratio between green and red cells so I could come up ratio of apoptotic cells in different treatment group.
I’m not sure what false-color and gray-color is. Could you please tell me how to check this. Thank you

Thanks,
Dinh


#4

When you open your image files, do they appear in shades of gray, or are the green cells colored green (and red cells colored red)?


#5

Hi Martha, the green cells appear green stained and the red cells appear red stain.


#6

So, first, you will need to either export your images so they appear in grayscale; or you can try the example pipeline, ColorToGray. Then, once you have your grayscale images saved, you can start working on your pipeline. What you will need some of the following modules:
LoadImages
CorrectIllumination_Calculate (two of these-- one for the green channel and one for the red channel)
CorrectIllumination_Apply (two of these-- one for the green channel and one for the red channel)
IdentifyPrimAutomatic (to identify green cells)
IdentifyPrimAutomatic (to identify red cells)
MeasureObjectIntensity (same comment as above… for the measure modules)
MeasureObjectIntensity
CalculateRatios
ExportToExcel

You might need other modules, depending on whether you decide to measure different features of the images (ie: Texture, Area, etc). The ‘help’ for each module will be a good reference for setting up the parameters.

If you would like to see how some of the modules work when identifying or measuring object, be sure to download a few of the examples and test them out.

Good luck,
martha


#7

Hi Martha,
I can convert from color to gray but i run into some problems. I don’t know how to use CorrectIllumination module

For CorrectIllumination_Calculate: What do i have to put for “image to be used to calculate the illumination function"
For CorrectIllumination_Apply: What do i have to put for “image to be corrected”, “call the corrected image”, “call the illumination correction function image to be used to carry out the correction”, “how do you want to apply the illumination correction function?”, and " Choose rescaling method?”


#8

Hi,
We have a tutorial, which explains various questions about the CorrectIllumination modules. Please refer to cellprofiler.org/linked_files/Pa … s_2008.pdf …The example that corresponds with the PDF is called, “ClassifiedColonies”.


#9

I got a problem when I run the image analyze. I don’t know what is wrong with my IdentifyPrimAutomatic. Here is my IdentifyPrimeAutomatic setting:
CorrRed, Type of diameter: 10,40 (how do i figure the real pixel for my cells), MoG Global

There was a problem running the analysis module IdentifyPrimAutomatic which is number 10. Error using ==> cat
Out of memory. Type HELP MEMORY for your options.

Stack:
IdentifyPrimAutomatic in C:\Documents and Settings\bwong\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\Modules\IdentifyPrimAutomatic.m (1017)
AnalyzeImagesButton_Callback in C:\Documents and Settings\bwong\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (9409)
gui_mainfcn in C:\Documents and Settings\bwong\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (11138)
CellProfiler in C:\Documents and Settings\bwong\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (55)


#10

It looks like your computer does not have enough memory. The ‘Out of Memory’ issues appear when the pipeline is running a lot of cycles, or your image is very large and you are trying to identify objects that are very small. The first issue can be solved by changing your preferences to either select certain figure windows or to not show any figure windows. The latter problem can be adjusted by measuring the actual diameter of your objects. To do this, you can double-click on the image filename (in the lower left box in the CellProfiler window), click on '‘CellProfiler Image Tools’>>‘Show or Hide Pixel Data’. Of course, if your diameter size is correct, then it might be worth trying to resize your image using the Resize module.

~martha


#11

The problem about memory gone. But I run into another problem. Here is the message:


There was a problem running the image analysis. Sorry, it is unclear what the problem is. It would be wise to close the entire CellProfiler program in case something strange has happened to the settings. The output file may be unreliable as well. Matlab says the error is: Invalid field name: ‘UneditedSegmentedRed Flourescen’. in the IdentifyPrimAutomatic module, which is module #10 in the pipeline.

Stack:
IdentifyPrimAutomatic in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\Modules\IdentifyPrimAutomatic.m (1101)
AnalyzeImagesButton_Callback in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (9409)
gui_mainfcn in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (11138)
CellProfiler in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (55)


#12

Try removing the space in “UneditedSegmentedRed Flourescen” for your object name.


#13

I could be able to run analyze image. However, I got zero Red cells and Green cells for my result. Could you please help me with this problem? Thank you.


#14

Well, you will need to adjust the settings (based on the objects that you are trying to identify). For a detailed explanation of how the IdentifyPrimAutomatic module works, please read the Current Protocols for Molecular Biology book chapter that is linked on the CellProfiler.org/examples.htm page.


#15

I used CellProfiler to measure my object diameter. My cells aren’t round. They has neuron-like shape. So I decided to measure the length. They range from 10 to 100. So I put that into IdentifyPrimAutomatic. It sill doesn’t give me any cell. I’m not sure what I did wrong here. Please give me some help. Thanks.
By the way, I couldn’t find any protocol on Molecular Biology on Example page. Coudd you send me the link? Thank you. Sorry for bothering you so much. I really appreciate your help.


#16

Here is the link to the PDF:
cellprofiler.org/linked_files/Pa … s_2008.pdf

Also, you might want to try downloading a few examples (ie: the ‘Fruit fly cells’ or ‘Speckle counting’) to get an idea of how IdentifyPrimAutomatic works. It is possible that the lower threshold is set incorrectly, or if the thresholding method (ie: Otsu Global, etc) is not the correct method for your objects. If, in the figure window for IdentifyPrimAutomatic, you see your objects outlined in red, then the size is incorrect and you can try changing the size.

good luck,
martha


#17

Here is another error with my CalculateRatio module:

There was a problem running the analysis module CalculateRatios which is number 10. Error using ==> rdivide
Matrix dimensions must agree.

Stack:
CalculateRatios in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\Modules\CalculateRatios.m (205)
AnalyzeImagesButton_Callback in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (9409)
gui_mainfcn in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (11138)
CellProfiler in C:\Documents and Settings\dlqnguyen\Desktop\CellProfiler_1.0.5122_XP32\CellProfiler_1.0.5122_XP32\CellProfiler_mcr\CellProfiler\CellProfiler.m (55)

Could you please help me troubleshoot for this problem?


#18

Sorry for the delay. CalculateRatios only works when you have the exact same number of objects that you are measuring and calculating ratios on. For example, if you have exactly 77 nuclei and 77 cytoplasms in the image, calculating ratios between them should work fine, but if you have 77 nuclei and 55 speckles it will not work so well.

Let us know if you are working with objects that meet these criteria and are still having troubles. Also be sure to get the latest version of CellProfiler, in case it really was a bug that’s been fixed by now.

Anne