Re: colocalization between confocal and two-photon: voxel matching

Posted by Arvydas Matiukas on
URL: http://confocal-microscopy-list.275.s1.nabble.com/xyzt-image-tp7580102p7580109.html

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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.: [phone]
fax: [phone]
email: [hidden email]