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Guy Cox-2 on
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It is more complicated than this. In MP mode you get the sqrt2 (1.414) resolution increase from the squaring of the PSF. In confocal at 1 Airy unit you don't get this. Therefore 800nm excitation in MP is equivalent to 565nm in confocal. (565 being the mean of excitation & emission). In practice, therefore, confocal and MP can be regarded as equivalent. So opening the pinhole for the confocal collection would not be a good idea - all it would do is confuse the colocalization by allowing contamination from out of focus light.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox 2nd edition, 2012 CRC Press
http://www.guycox.com/optical.htm______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Aust. Centre for Microscopy & Microanalysis, F09,
University of Sydney, NSW 2006
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-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Julio Vazquez
Sent: Thursday, 4 April 2013 5:13 AM
To:
[hidden email]
Subject: Re: colocalization between confocal and two-photon: voxel matching
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Hi Arvydas,
You want to keep your pinhole fully open for two-photon imaging..... in two photon mode, you are exciting one single spot in the specimen, so there is no need to have a pinhole to exclude out of focus light, since there isn't any. You want the pinhole open to maximize signal collection. Do not trust the estimate of the optical slice thickness in two photon mode that the LSM software gives you. The voxel size for two photon excitation is defined by the wavelength you use for excitation, I'm guessing around 800 nm, so the radius of your laser spot is only 1.6 times greater for DAPI compared to excitation with 488 nm. The thickness of the optical slice is fixed for two photon (size of exciting laser spot). You can open the pinhole a little bit for the green chanel to try to match (I'm guessing to maybe 1.5 Airy units), so this would give you voxels that are not so different.
Keep in mind that the accuracy of your colocalization will depend on other factors: how you threshold, how you subtract background, amount of noise in your images, etc.... If you are trying to "colocalize" structures that are significantly larger than the resolution of you microscope, the error contributed by the differences in voxel size will be comparatively small. If the structures you are comparing are near or below the resolution limit, then the error will get comparatively higher. As an extreme example, if you have two single molecules 100 nm apart, their images will mostly overlap and give a high degree of colocalization using conventional colocalization methods, while in reality the two molecules do not overlap at all. So my advice is to adjust teh pinhole for 488 to try to match the voxel size for DAPI as best as you can, and then use a sound colocalization method (and good controls) for your colocalization.
Deconvolving the data might be a good idea to reduce noise and tighten your PSFs before running your analysis.
I found the discussions on colocalization provided by SVI (scientific volume imaging) to be very useful. You can Google colocalization + SVI. to access their documentation.
Just saw Brian's response, so some of the above is redundant.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA
http://www.fhcrc.orgOn Apr 3, 2013, at 10:28 AM, Arvydas Matiukas wrote:
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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:
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