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The 2P may cause more background due to the broad excitation spectra exciting other molecules in the specimen -i.e. effectively more non specific label shows up. 

Cheers Mark

On 15/11/2013, at 2:00 pm, Laevsky, Gary S. <[log in to unmask]> wrote:

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> 
> Hi All,
> 
> I’m sure there is going to be a simple answer here, but it alludes me.
> 
> I know the psf is a function of NA and wavelength.
> 
> Obviously, with 1P we use a pinhole to exclude out of focus light, and when set at “1 Airy,” you have maximized the pinhole for the particular wavelength you are using.  With 2P, no pinhole is necessary because of the non-linear excitation mechanism.
> 
> A user just approached me and asked if/why there would be more out of focus light in a 2P image then in a 1P image with a properly set pinhole.  In a collaborators experiments, there seems to be more background in the 2P image (all other things equal).
> 
> Only thing I can think of is a poor beam profile on the 2P.  Maybe a large pulse width would excite a larger spot?
> 
> Thankful for the insight.
> 
> 
> Best,
> 
> Gary
> 
> 
> 
> Gary Laevsky, Ph.D.
> Confocal Imaging Facility Manager
> Dept. of Molecular Biology
> Washington Rd.
> Princeton University 
> Princeton, New Jersey, 08544-1014
> (O) 609 258 5432
> (C) 508 507 1310

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

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