"...the neurons also have different areas..."

That's the reason why ID yield higher values (even if they look less bright for the staining).

ImageJ manual:

1) Mean gray value (MGV) Average gray value within the selection. This is the sum of the gray values of all the pixels in the selection divided by the number of pixels. Reported in calibrated units (e.g., optical density) if Analyze▷Calibrate…↓ was used to calibrate the image. [...]

2) Integrated density (ID) The sum of the values of the pixels in the image or selection. This is equivalent to the product of Area and Mean Gray Value.

So

3) Mean Gray Value*Area = Integrated density

Please, take a look at this

https://www.researchgate.net/post/Mean_gray_intensity_value_or_the_integrated_density_value_which_among_the_two_value_is_more_appropriate_for_quantifying_the_fluorescence_image_data

Which method would be most appropriate for this knowing neurons vary in size amongst conditions?

I think you should go for average gray value, unless you want to make the point that, since neurons are bigger, they have a higher content of protein (which is usually not the case). To avoid the confounding factor of size, you should use AGV. You should probably report area and average gray values, that way ID is implicit in your results, but use agv for comparisons.

As a general advice, uploading pictures when you ask about this kind of things is usually a better way to get reliable and helpful answers ;)

Hope this helps,

Cheers,

J

J. Ramirez-Franco Thank you so much for your support. If using AGV do you recommend changing the image to greyscale? I have calculated the AGV without changing it (GFP protein channel) before, not sure if this would be the same?

To change to greyscale by going to IMage-> Type--> 8-bit. If so, do they modify the data/pixel value?

Hi,

Absolutely. Measurements should be performed in grayscale (8bit or 16bit) images.

Anyway, for getting help with this type of questions is always convenient to share a sample image explaining what you have done so far over the image and what you want to achieve.

To change to greyscale by going to IMage-> Type--> 8-bit. If so, do they modify the data/pixel value?

The truth is I cannot answer your question without knowing about your original images (are them RGB, composites, 16bits, colored 8bits, ...?).

To answer this question try to read and understand these links:

https://imagej.net/imaging/color-image-processing

https://imagej.nih.gov/ij/docs/menus/image.html#type

Cheers,

J

J. Ramirez-Franco. Thank you so much for your fast replies. My images are already at 8-bit. They are coloured. GFP channel. They are quite dark when I open them on image J- this is because of the laser intensity on Zeiss that I wanted to moderate and I had to adjust brightness and contrast to decrease saturation of pixels.

I am attaching an example, but I had to increase the brightness & adjust the contrast so that u can see.

When I quantify them I normally increase the brightness/contrast without pressing 'apply' this allows me to identify where the ROI are without affecting my pixel values