Posted by
Armstrong, Brian on
URL: http://confocal-microscopy-list.275.s1.nabble.com/xyzt-image-tp7580102p7580110.html
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Hello, when performing 2-Photon imaging keep the pinhole at maximum. In 2PE an optical slice is created via pinpoint excitation and not through the pinhole in the emission path (as is the case for Confocal imaging). The optical slice may be smaller/thinner in the 2P case. There is extensive explanation of this in Pawley's book (Handbook of Biological Confocal Microscopy) and in Diaspro's book (Confocal and 2-Photon Microscopy, Foundations Applications and Advances, Alberto Diaspro.
Moreover, I have performed this same analysis on this same instrument (in press) so I may be able to answer other questions.
Cheers,
Brian D Armstrong PhD
Assistant Research Professor
Director, Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Arvydas Matiukas
Sent: Wednesday, April 03, 2013 10:29 AM
To:
[hidden email]
Subject: Re: colocalization between confocal and two-photon: voxel matching
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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.: 315-464-7997
fax: 315-464-8014
email:
[hidden email]
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