The confocal psf is not the product of excitation and emission psfs unless the pinhole is very small (much less than 1 Airy unit). In normal use (pinhole at 1 Airy) it is just the excitation psf. So it will actually be quite similar to the 2p one. (At 800nm Rayleigh resolution is ~350nm and 350/1.4 gives ~250nm.)
I agree with an earlier correspondent that the greater background is almost certainly due to the fact that 2p excitation spectra tend to be broader than 1p ones. If so it should be possible eliminate it by a more selective detection filter.
Guy
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Jeff Reece
Sent: Sunday, 17 November 2013 1:13 AM
To: [log in to unmask]
Subject: Re: 2P vs 1P psf
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First I will assume that "all other things equal" means that the 2P and 1P images have the same SNR. (A lower SNR will give you a lower effective resolution.)
If you are within a few microns of the cover glass, resolution should be worse with 2P than 1P. This is because the much longer excitation wavelength in 2P creates a larger psf. Even though the effective psf in 2P mode is the square of the raw excitation psf, the confocal psf is the product of the excitation and emissions psfs.
Then, as you get away from the cover glass, at some point-- the resolution in the 2P image will start to look better than 1P. This is because the shorter excitation wavelength in 1P has a harder time focusing at the deeper depths, due to the difference between immersion media RI and sample RI, and/or the dispersion of the sample as a function of wavelength. Exactly where the transition occurs depends on these factors, maybe others that have escaped me at the moment. So I say "a few microns" but it is more likely in the tens of microns when using water immersion with live sample.
Since your user is in the realm where 1P looks better then 2P, then it is likely that there is also not much dispersion of the emission through the sample, so one could collect the 2P emission on the descanned detector with the pinhole closed down (to a little less than 1AU based on the 2P psf), without losing much signal from the focal plane. Resolution in 2P could then be increased, with the added benefit of removing other background from OOF planes. Of course this doesn't work in thick tissue where there is dispersion.
If the 2P "background" signal you mention is not due to nearby OOF signal, but seems ever-present and non-changing as you move around a sparsely stained sample in xy:
2P requires a lot more laser power at the sample than 1P to get the same signal back; the extra power density in the focal volume is necessary in order for 2P to
occur. Thus reflection of the laser on all optical interfaces, relative to the fluorescence signal, is much higher with 2P than 1P at the point where the emission hits the dichroic. So the dichroic and emission filter need to be better at blocking the laser for 2P than 1P.
If you have this problem, then obviously the best fix is to replace or double up the emission filter so you get better blocking of the laser. If your user is in the ("thin sample") case mentioned above, then you could alternatively use the quick fix of sending the 2P emission to the descanned detector and closing down the pinhole.
I hope my lengthy explanation has helped.
Cheers,
Jeff
>________________________________
> From: "Laevsky, Gary S." <[log in to unmask]>
>To: [log in to unmask]
>Sent: Friday, November 15, 2013 9:00 AM
>Subject: 2P vs 1P psf
>
>
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the confocal microscopy listserv, go to:
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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
>
>
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