Calculate three specific color intensities

Hello all,

You all are the experts, and I wish to ask you about image analysis to aid my pursuit for scientific truth. My issue seems simple but I think I am beginning to understand it may be complicated?

My goal: I would like to calculate stain intensities for all three channels (RGB) separately for a SINGLE selection. That is, I need to be able to make a single selection which is the exact same across all three channels, and analyze the respective intensities for it (i.e. red intensity, green intensity, blue intensity).

Is this possible for me to do and how would I go about producing these results?

Thank you very much in advance for your help on this matter.

Hi Kev

To do this using the interface is pretty simple. Once you have your selection in the ROIManager (ctrl + t) try the multi-measure option under the “more” menu of the ROIManager. Set what you want to measure using Analyse>Set Measurements.

Best
Jeremy

Hi Jeremy,

Thank you for your reply. However, I’m unsure if it is what I’m looking for. I still only get a single integrated density for a single selection. What I need is, from a single selection, to produce a red-only integrated density, a green-only integrated density, and a blue-only integrated density.

Maybe I’m just not quite understanding your suggestion, if your suggestion in fact produces that result?

Thank you again for your help.

@Kev No worries, you should get three rows of measurements (for red, green and blue). If you have an RGB image try converting it to a composite (Image > Color > Make Composite). When you run multi-measure you should then see a check box “measure all three slices”. Make sure this is ticked.

Jeremy

Good day Kev,

here is a stand-alone macro that should do what you need:

run( “Clown (14K)” );
run( “Make Composite” );
makeOval( 12, 12, 100, 100 );
roiManager( “Add” );
roiManager( “multi-measure measure_all one append” );

Paste the code to an empty macro text window:
Plugins >> New >> Macro
(You need an internet connection.)

Regards

Herbie

Thank you both for your kind suggestions. They both work splendidly for my images. If you don’t mind, may I ask you a follow-up question? If not, I can make a new topic, no problem.

With your help, I’ve solved how to measure separate RGB intensities using the same selection. My next question relates to making an actual selection. The purpose of what I am doing is, using skeletal muscle stained for three differing myosin heavy chain isoforms, to quantify fiber type and area based on the relative intensities of each isoform (e.g. if a fiber has 75% red, 20% green, and 5% blue- I would quantify that fiber as a predominantly ‘red’ phenotype).

My ‘issue’ is the lack of consistency I am able to produce by manual drawing of the fibers. I have played around with threshold function a bit. However, it does not seem to truly ‘get the job done’

Do you guys have any advice as to how I can more reliably make the area drawing, then use the area drawing to measure size and relative intensities?

Thank you. I am attaching a sample image for reference. An accessory problem is the lack of membrane stain for our samples. However, we are unable to run the samples again.

Original TIF file: download

Kev,

although there might be a chance to segment your images by traditional image processing techniques, I would try a classifier. Look for WEKA which comes with Fiji.

Good luck

Herbie

1 Like

Thank you Herbie, I appreciate the suggestion. I have downloaded Fiji and am working with it now.

I have also posted this new question as a separate topic, as I want to gather what all my options are. The goal in mind being to use the best and most accurate way to answer my research question.

Again, thank you for all of your help. I really do appreciate it.