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Well, you would, obviously.  Why would you use 2P in descanned mode?  That is nonsensical.  

			Guy                                                 


-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Arvydas Matiukas
Sent: Saturday, 16 November 2013 8:38 AM
To: [log in to unmask]
Subject: Re: 2P vs 1P psf

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I  typically notice more background in 2P mode (descanned detector) vs
1P
for our LSM510 NLO. Unless the sample is thick I suggest users to
employ 1P mode.
 
Best,
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: [log in to unmask]
>>> Mark Cannell <[log in to unmask]> 11/15/2013 9:07 AM >>>
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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:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
> 
> 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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