Posted by
phil laissue-2 on
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Hi Arvydas,
further to the other two replies, I'll just add a few references for
quantifying colocalisation. By no means a comprehensive list, but in my
humble opinion some of the most user-friendly discussions/approaches.
Really depends on the structures in question, there's not one single
approach that works best.
pixel-based:
http://www.ncbi.nlm.nih.gov/pubmed/23026999diva-portal.org/smash/get/diva2:563664/FULLTEXT02
coloc2:
http://fiji.sc/Colocalization_Analysishttp://www.ncbi.nlm.nih.gov/pubmed/21209361http://www.ncbi.nlm.nih.gov/pubmed/15189895object-based:
http://www.ncbi.nlm.nih.gov/pubmed/20858446http://crg.ubc.ca/moore/http://www.ncbi.nlm.nih.gov/pubmed/19746416http://www.ncbi.nlm.nih.gov/pubmed/23381680(happy to send you reprint and matlab code)
Also worth checking out:
http://www.ncbi.nlm.nih.gov/pubmed/17210054http://www.ncbi.nlm.nih.gov/pubmed/22086768Hope this helps. Kind regards
Philippe
____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue <
http://privatewww.essex.ac.uk/%7Eplaissue>
_____________________________________
Philippe Laissue, PhD, Bioimaging Manager
School of Biological Sciences, Room 4.17
University of Essex, Colchester CO4 3SQ, UK
(0044) 01206 872246 / (0044) 07842 676 456
[hidden email]
privatewww.essex.ac.uk/~plaissue
On Wed, Apr 3, 2013 at 6:28 PM, Arvydas Matiukas <
[hidden email]>wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hello list,
>
>
> One of my Core users wants to measure colocalization between
> DAPI labeled and FITC-immunolabeled structures in nuclei of
> her fixed cells. Visual inspection shows good level of colocalization.
>
> However, what would be the correct way to quantify it.
> Our confocal is LSM510 META NLO. No UV or 405nm laser
> so two-photon mode at 780nm is used to excite blue dye
> while FITC is excited at 488nm in confocal mode . At default
> pinhole settings (1 airy unit for confocal and max=~10 A.u.
> for two-photon) both blue and green signals are good
> but the optical slices (and voxel sizes) differ by the factor of 10.
> Letting the software (LSM) to equalize the slices at 0.6um reduces the
> pinhole to 0.76 for confocal and 1.0 for two-photon.
> Unfortunately at such small pinhole for two-photon the
> blue signal is lost in noise (even at 5% laser power and 16 times
> averaging, further increase may bleach the dye during 15 min
> long scan). I can get the blue signal back to a measurable level
> by increasing the two-photon pinhole to 4 A.u. but this creates
> a four-fold mismatch in blue/green voxel size. I could boost the
> blue signal by increasing pixel size but the loss in resolution
> is not desirable.
>
> My first thought was to switch to red DNA stain and then
> do colocalization between two confocal signals with identical
> voxels. However, the user already completed experiment
> using DAPI staining, and it worked well. My question is
> what would be the correct way to acquire the confocal/two-photon
> z-stacks and subsequently quantify the colocalization.
> And related question if reducing pinhole works the same
> way for two-photon as for a regular confocal.
>
> Any feedback/advice/idea are very welcome,
> Arvydas
> ----------------------------
>
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Core
> Department of Neurosci& Physiology
> SUNY Upstate Medical University
> 766 Irving Ave., WH 3167
> Syracuse, NY 13210
> tel.: 315-464-7997
> fax: 315-464-8014
> email:
[hidden email]
>