Endosomal detection

[Ksenia Oguievetskaia]

Hi,

     

I am a new Icy user and I have a lot of questions... 

I am looking for a colocalisation of two proteins on endosomes, that is partial.. but I have quite noisy images because of a cytoplasmic presence of those. 

              

My idea is to 'extract' the endosomal staining and to quantify the colocalisation. 

For this I am trying to use the Spot Detector and I have troubles to detect all the endosomes... 

Is it possible to get some help for the analysis? Can I send you one image? 

             

The images are from a spinning disk, they are deconvoluted. They come from movies and Ideally I would like to track the extracted endosomes in time ..   

                   

Thanks in advance!

Ksenia

[Stephane]

Hi Ksenia,

Indeed it would be nice to have a test image so we could try to help you with your analysis :)
You can attach image to message so don't hesitate !

Best,

- Stephane

[Ksenia Oguievetskaia]

Hi Stephane,

 

thank you for the rapid answer. 

I attach here two pictures, Z stacks.

 

Thank you for the feed back.. 

Ksenia

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Ksenia

          

[Stephane]

Hi,

Thanks for the pictures, indeed the pictures are very noisy.

I obtain i believe acceptable results on the 561 image with the following parameters in the spot detector :
 - detect bright spot
 - force use of 2D wavelet for 3D (you need more Z slices for real 3D detection).
 - scale 2 with 18 sensitivity

But the 491 channel image is too noisy and you can't really tell where is the protein and where is the noise :-/
Normally that is the good method to perform colocalization (after the detection you should use the Colocalization Studio plugin to get the coloc statistics).
Do you have the raw images (without deconvolution) ?

Best,

- Stephane

[Ksenia Oguievetskaia]

Hi,  

 

it happens to be my protein of interest in the 491 channel :/ 

that is our problem, but it is the best we can get. Here are two not deconvoluted stacks at two time points: 

https://drive.google.com/file/d/0B0QeBfOPArb0WjRiN3huQ3A0dHc/view?usp=sharing

the protein of interest is located on the moving vesicles which allows us to conclude that the staining is spécific. We also know that this protein is cytoplasmic which gives the background.. 

          

thanks 

best

ksenia

[Stephane]

Hi,

I tried to do a mean Z projection and using the Spot Detection afterward.. it does help. But i guess you cannot use it as you loss the Z spacial colocalization information.
Honestly I'm not sure which are the best image to perform the analysis... in both case it's quite noisy. If you lower the spot detector sensitivity you will only grab the brighter spots (good confidence about them). If you have many images to analyses it may give you some results... i don't think you can do much more than trying to play with the spot detector sensitivity parameters to find the best compromise for that type of image.

- Stephane