If you are analyzing 16-bit stacks, the auto-threhold routine in JaCoP will be very slow. If you are using auto-thresholding, try changing images to 8-bit first.
> Hi Paul,
>
> I will not comment on principles of colocalisation, as I not competent
> enough and there better people to answer that.
> I just wanted to point out that one reason you may find the JACoP so slow
> is that you may be running too many analyses in it. I have found that by
> selecting which parameters I really needed, there was little difference
> between the two.
> Depending on your data, the JACoP also offers the possibility to adjust the
> thresholds manually, which in some situations may be more important (if
> matching intensities in the pixels where correlation is analysed are not of
> primary interest, but objects are).
>
> Again, there are lots of knowledgeable people in the community.
> For a good overview of colocalisation analysis principles, I found the
> review by Bolte and Cordelière (J Microscopy 2006) most helpful. You may
> already have read it, in which case good luck!
>
>
> regards
> Sonia Fargue
>
>
> 2013/9/19 Paul Buchanan <
[hidden email]>
>
>> Hi All,
>>
>> Hoping you can give me a few pointers with regards to co-localisation in
>> image j and FiJi.
>>
>> Firstly can some one explain to me in a bit more about intensity based
>> co-localisation. To generate a histogram from which the correlation
>> co-efficient can be generated, the intensity of one can is plotted against
>> the other. But how does this take into account the location of the pixels?
>> Sure this method just tells you if pixels in one channel have a similar
>> intensity to another channel? How does that describe co-localisation. I
>> have read all the papers referenced for the plug ins but just missing out
>> on a few basic principles.
>>
>> I have been using coloc2 and JaCop to look at receptor co-localisation
>> with various endsomes as it traffics through the cell. Firstly i have been
>> finding JaCoP extremely slow, so slow i cant run it with my images. Ok my
>> images are ~200mb each and have about 20 slices in each but coloc2 runs
>> through in about 5 - 10 minutes. I have been getting some r values and they
>> seem to represent what i see in the overlay images with regards to more or
>> less yellow colour. But i have been asked when explaining my work what
>> exactly the Pearsons coefficient shows and as stated above am unsure if it
>> represents location of co-localisation as well as the intensity. Also am
>> not sure if this r value represents the whole z stack i have imaged? and
>> what what about slices that have no co-localisation due to being above or
>> below the cell does this on skew the results.
>>
>> I have been looking into doing some object based colocalisation as it
>> seems like it would be a better method to confirm co localisation over my z
>> stacks but again jacop takes ages to do anything.
>>
>> Any advice would be gratefully appreciated.
>>
>> Thanks
>>
>> Paul Buchanan
>>
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