measurement median intensity and setup "intensity ratio options" button

[marcio0314]

Hello,

I would like to measure the average median intensity, is it possible to do this?

and another question is when I to mark the box in the "S" button:

• use filter on nuclei image radius filter
• remove scale what are the functions of that options?

[marcio0314]

@volker-baecker and thanks in advance for your help

[marcio0314]

Hello @volker-baecker. Can you help me?

Thanks

[volker-baecker]

Hi @marcio0314, sorry for the late reply, I've been on holiday.

The Intensity Ratio Nuclei Cytoplasm Tool measures the mean intensity in the nuclei and in the cytoplasm. It does not measure the median of the intensity values. I don't know what you mean by average median, please explain.

"use filter on nuclei image radius filter": If you select the "use filter on nuclei image" option, a Gaussian-blur filter with the sigma you enter in the radius field (it's named radius for historical reasons, I should rename it from radius to sigma now) will be applied to the nuclei-image, before the nuclei are segmented.

The "remove scale" option will remove the spatial calibration of the image if any, so that the "min. nucleus area" and the area measurements are in pixel.

Best, Volker

[marcio0314]

HI @volker-baecker ,

is possible to measure median? I just need the median value from Cytoplasm and nuclei.

Average median means the average of median cytoplasm or nuclei. Data results after analysis show me total intensity. but don't mind.

Thanks, Marcio

[volker-baecker]

Hi @marcio0314, I added the nuclei and cytoplasm median to the results. It is the value after background correction. There is one median for all nuclei in one image and one median for the whole cytoplasm in one image. If that seems useful to you, please download the latest version from Intensity Ratio Nuclei Cytoplasm Tool. Best, Volker

[marcio0314]

Hi @volker-baecker ,

Thanks a lot!! I will try the new macro update.

Best, Marcio

[marcio0314]

Hi @volker-baecker

the latest version is not working.

1. when I use the analysis in a single image, the results don't have the median intensity.
2. 2. when I use the analysis to a batch mode do not work. program show me a error message about missing some file (picture attached)

Best, Marcio

[volker-baecker]

It works on a single image for me, as you can see in the screenshot. I'll try the batch processing.

[volker-baecker]

The batch processing also works on my data

If you can send me some of your images, I can probably find out what's happening.

Best, Volker

[marcio0314]

Hi @volker-baecker

I made the process, but I don't know why I got wrong median fluorescence intensities values (like lower integer number as 2, 3, 4) don't make sense for me. I mean, I understand that values of median fluorescence intensity are since 0 to ∞ and always are higher values....

I attached some images.

[volker-baecker]

Hi @marcio0314, the median is the value in the middle of the list of sorted intensity values (the average of the two middle values for an even number of elements). Generally there is no reason for it to be a "higher" value. If most of the values are "lower" values the median will also have a "lower" value.

As I said before the mean and median values are calculated after background subtraction. Therefore the values will be lower compared to the intensity values in the input images. Looking at your images the low values are no surprise, since there are a lot of nuclei without any signal in the other channel. In fact the tool was not made for a situation where there are nuclei that do not have a staining in the cytoplasm channel. Please make sure that the results would make sense for your biological question.

Here are the results I get using the default parameter values (except for the channel names):

[marcio0314]

Hi @volker-baecker ,

I see... I have a problem to express in all cells the Fluorescence. this fluorescence is to measure nucleus/ cytoplasm translocation of fluorescent protein, and the nucleus is stained with DAPI. is there some way to choose only cells that have the red fluorescence and remove nuclei that not have a co-occurrence by both fluorescent reporters?

Thanks, Marcio

[volker-baecker]

Hi @marcio0314,

it's surly possible to write a macro that does this, but it goes beyond the scope of this tool. The advantage of this tool is its simplicity. It segments the nuclei, without separating them and subtracts the background. All pixels belonging neither to the background nor to the nuclei are considered cytoplasm. If you can segment the individual cells you would probably prefer to measure the intensity ratio on a cell by cell basis.

Best, Volker