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Hi Gary,
Is your collaborator using a non-descanned detector? A computer monitor
near the scope could produce enough background light to interfere with his
imaging. Those pick up stray light like a widefield scope does, sometimes
worse.
All the best,
TF
Timothy Feinstein, Ph.D. | Confocal Manager
333 Bostwick Ave., N.E., Grand Rapids, Michigan 49503
Phone: 616-234-5819 | Email: [log in to unmask]
On 11/15/13, 9:00 AM, "Laevsky, Gary S." <[log in to unmask]> wrote:
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>
>Hi All,
>
>Iım 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 ³1 Airy,² 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
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