Coloc2: calculating the approximate PSF



Hi Stephanmg,

I am also analizing some images using Coloc2 but I don’t know how calculate the approximate PSF. It would be nice if you could tell me how can I do it.

Thank you very much.


Colocalization analysis


This is info I got from the Coloc2 expert @chalkie666… If images are collected properly the PSF should be close to 3 pixels. To be sure, you can calculate it knowing the optical resolution d = lambda / 2*NA. (NA is the second number written on the objective lens, after the slash after the magnification number.) Then divide d by the pixel spacing in the image and that is the approximate PSF size.

You could also use this tool from SVI: Nyquist Calculator… as ultimately, the psf depends on many factors: the magnification, objective lens, camera binning, immersion oil, mounting medium, …

eta :slight_smile:


Hello Etarena,

Thank you so much for the info. I still have some questions, it would be nice if you could help me:

  1. Is lambda the value of the laser wavelength???
  2. If I use the Nyquist Calculator I do not obtain a value for the PSF, I obtain 3 values in X, Y and Z, Which of these values ​​is the one that I have to use as the value of the PSF in coloc2?

Thank you very much and sorry for the inconveniences,



Hello Etarena,

This is Alfonsa again, I forgot asked you if the “pixel spacing” is the “pixel size”…

Thank tou so much


Good day,

there is no pixel size but only a pixel spacing however, because this fact appears to be a big secret, most often both expressions are used in the same sense.




Hi Herbie,

Thank you!!!




Yes. :slight_smile:

What are the values that you calculate in the end? Can you provide me with an example image so I can take a look myself (make sure it’s in the original formate)?

If you are worried - you can just use @chalkie666’s calculation and you will be fine. :slight_smile: But the size that you put in for the PSF is in pixels… so keep that in mind.



Good morning Etarena,

I’m sorry to bother you and I thank you very much for your help.
I attach the image of a bead, of which I am trying to calculate the value of the PSF to put it in the “PSF” section of the plugin Coloc2.
I did:
d = lambda / 2*NA;
Then I divided d by the pixel spacing (0.041um = 41nm);
d/pixel spacing = 174.2/41 = 4.2.
PSF = 4.2

Using the Nyquist Calculator I obtain 3 values

as you can observe in this image I obtain 3 values x: 43nm, y: 43nm, z: 130nm; and I don’t know which value is the PSF value I need to Coloc 2.

thank you very much for helping me and sorry for the inconvenience.



here goes the bead image (It looks like I forgot sending it in my previous post)



Looks like your magic number is 4 !! :slight_smile:



Hello all,

yes I used this calculator too.



Just to be sure, Coloc2 says:
PSF has to be provided in pixels of my image, correct?

The Nyquist calculator gives in nm:
x: 247 nm
y: 247 nm
z: 3889 nm

Thus if I know my pixel spacing d I can divide by d resulting in the value I’ve to put into Coloc2.

Best wishes,