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2P vs 1P psf

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Laevsky, Gary S.

2P vs 1P psf

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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 Cannell-2

Re: 2P vs 1P psf

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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. <[hidden email]> 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

[hidden email]

Feinstein, Timothy

Re: 2P vs 1P psf

In reply to this post by Laevsky, Gary S.

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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: [hidden email]

On 11/15/13, 9:00 AM, "Laevsky, Gary S." <[hidden email]> wrote:

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://scanmail.trustwave.com/?c=129&d=rqmG0jAq5wSLcDwzveugGEfcTBlOgb6VWnC
>SP5ImQw&u=http%3a%2f%2flists%2eumn%2eedu%2fcgi-bin%2fwa%3fA0%3dconfocalmic
>roscopy
>*****
>
>Hi All,
>
>I¹m 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

Arvydas Matiukas

Re: 2P vs 1P psf

In reply to this post by Mark Cannell-2

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

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: [hidden email]
>>> Mark Cannell <[hidden email]> 11/15/2013 9:07 AM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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. <[hidden email]>
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

[hidden email]

Guy Cox-2

Re: 2P vs 1P psf

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

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

Guy

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

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

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: [hidden email]
>>> Mark Cannell <[hidden email]> 11/15/2013 9:07 AM >>>
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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. <[hidden email]>
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?

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

[hidden email]

Jeff Reece

Re: 2P vs 1P psf

In reply to this post by Laevsky, Gary S.

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

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." <[hidden email]>
>To: [hidden email]
>Sent: Friday, November 15, 2013 9:00 AM
>Subject: 2P vs 1P psf
>
>
>*****
>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
>
>

Guy Cox-2

Re: 2P vs 1P psf

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:[hidden email]] On Behalf Of Jeff Reece
Sent: Sunday, 17 November 2013 1:13 AM
To: [hidden email]
Subject: Re: 2P vs 1P psf

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

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." <[hidden email]>
>To: [hidden email]
>Sent: Friday, November 15, 2013 9:00 AM
>Subject: 2P vs 1P psf
>
>
>*****
>To join, leave or search
the confocal microscopy listserv, go to:

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 Cannell-2

Re: 2P vs 1P psf

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

It’s quite easy to check for laser excitation getting past the filters. Just run the laser in CW (stop mode locking). In my experience interference filters seem to block the IR very well.

HTH

Mark

>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeff Reece
> Sent: Sunday, 17 November 2013 1:13 AM
> To: [hidden email]
> Subject: Re: 2P vs 1P psf
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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." <[hidden email]>
>> To: [hidden email]
>> Sent: Friday, November 15, 2013 9:00 AM
>> Subject: 2P vs 1P psf
>>
>>
>> *****
>> 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

[hidden email]

Jeff Reece

Re: 2P vs 1P psf

In reply to this post by Guy Cox-2

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

Is there a reference for the confocal psf being equal to the excitation psf when the pinhole=1AU (free access via the web is requested). It is not intuitive that the confocal psf would leave out the optics (pinhole) of the emission path.

Thanks,
Jeff

>________________________________
> From: Guy Cox <[hidden email]>
>To: [hidden email]
>Sent: Sunday, November 17, 2013 1:18 AM
>Subject: Re: 2P vs 1P psf
>
>
>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:[hidden email]] On Behalf Of Jeff Reece
>Sent: Sunday, 17 November 2013 1:13 AM
>To: [hidden email]
>Subject: Re: 2P vs 1P psf
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>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." <[hidden email]>
>>To: [hidden email]
>>Sent: Friday, November 15, 2013 9:00 AM
>>Subject: 2P vs 1P psf
>>
>>
>>*****
>>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
>>
>>
>
>
>

Lutz Schaefer

Re: 2P vs 1P psf

In reply to this post by Guy Cox-2

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

Guy,
in any incoherent modality, the image formation is always composed out of
the product of emission and excitation psf's. In the special case of point
scanning confocal microscopy, its only that the (widefield) emission psf is
further convolved by the geometry of the pinhole before multiplication.
Therefore, of course you are right in saying that towards a more open
pinhole the emission psf component approaches a constant. In 2p, the
excitation psf is significantly smaller (approaching the square with no
aberrations present) and in theory a pinhole in the emission path could
further increase the sectioning resolution. However, numerically this is
insignificant as due to the square, the excitation psf already is much
smaller than the emission psf could possibly become, even with pinhole. On
top of all the often very low 2p SNR and present SA forbids to observe
minute improvements.

Regards
Lutz

-----Original Message-----
From: Guy Cox
Sent: Sunday, November 17, 2013 1:18 AM
To: [hidden email]
Subject: Re: 2P vs 1P psf

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:[hidden email]] On
Behalf Of Jeff Reece
Sent: Sunday, 17 November 2013 1:13 AM
To: [hidden email]
Subject: Re: 2P vs 1P psf

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

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." <[hidden email]>
>To: [hidden email]
>Sent: Friday, November 15, 2013 9:00 AM
>Subject: 2P vs 1P psf
>
>
>*****
>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
>
>

mmodel

Re: 2P vs 1P psf

In reply to this post by Jeff Reece

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

My understanding of it is not terribly scientific, but here's how I reason.

1. First, suppose we are looking at a self-luminous point with a confocal microscope. The pinhole will just scan the image plane and, one step at a time, will collect the same Airy pattern as a planar detector would:
I = PSF(det).

2. Next, suppose we are looking at a fluorescent point. In confocal scanning, the illumination is not uniform but has its own Airy shape, PSF(ill). That means that when the pinhole is moved on the image plane away from the image of the point, the illumination (which is conjugate to the pinhole) becomes reduced: the laser is focused away from the point, which is now illuminated only by diffraction rings of PSF(Ill). So compared to the previous situation, we have an additional factor PSF(Ill,) and the total intensity distribution becomes

I = PSF(det)*PSF(Ill)

3. And, additionally, if you do scanning without a pinhole then

I = PSF(Ill)

compared to PSF(det) in wide field
----
Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Jeff Reece [[hidden email]]
Sent: Sunday, November 17, 2013 10:06 AM
To: [hidden email]
Subject: Re: 2P vs 1P psf

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

Is there a reference for the confocal psf being equal to the excitation psf when the pinhole=1AU (free access via the web is requested). It is not intuitive that the confocal psf would leave out the optics (pinhole) of the emission path.

Thanks,
Jeff

Lutz Schaefer

Re: 2P vs 1P psf

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

Michael,
you are correct, but the illumination PSF should dominate in the product (2)
as its support volume is much smaller. To be exact, it can be ideally up to
PSF(ill)^2, using the standard vectorial models (Egner & Hell et. al,
somewhere in Jim's Confocal Handbook). Then, in the product with the larger
PSF(det) (including the convolution, even with an infinitely small pinhole)
leaves you essentially still approximately with PSF(ill)^2. Unless you have
spherical aberrations to the extend that the "inferred illumination spot"
(meaning the effective volume of 2p generation) is much larger, you would
not be able to see a difference. Unfortunately the nonlinearity (modeled
with ^2) really only lets 2p generation happen at a high intensity
threshold, which in turn gives you the small spot...

Lutz

-----Original Message-----
From: MODEL, MICHAEL
Sent: Sunday, November 17, 2013 3:00 PM
To: [hidden email]
Subject: Re: 2P vs 1P psf

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

My understanding of it is not terribly scientific, but here's how I reason.

1. First, suppose we are looking at a self-luminous point with a confocal
microscope. The pinhole will just scan the image plane and, one step at a
time, will collect the same Airy pattern as a planar detector would:
I = PSF(det).

2. Next, suppose we are looking at a fluorescent point. In confocal
scanning, the illumination is not uniform but has its own Airy shape,
PSF(ill). That means that when the pinhole is moved on the image plane away
from the image of the point, the illumination (which is conjugate to the
pinhole) becomes reduced: the laser is focused away from the point, which is
now illuminated only by diffraction rings of PSF(Ill). So compared to the
previous situation, we have an additional factor PSF(Ill,) and the total
intensity distribution becomes

I = PSF(det)*PSF(Ill)

3. And, additionally, if you do scanning without a pinhole then

I = PSF(Ill)

compared to PSF(det) in wide field
----
Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf
of Jeff Reece [[hidden email]]
Sent: Sunday, November 17, 2013 10:06 AM
To: [hidden email]
Subject: Re: 2P vs 1P psf

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

Is there a reference for the confocal psf being equal to the excitation psf
when the pinhole=1AU (free access via the web is requested). It is not
intuitive that the confocal psf would leave out the optics (pinhole) of the
emission path.

Thanks,
Jeff

>________________________________
> From: Guy Cox <[hidden email]>
>To: [hidden email]
>Sent: Sunday, November 17, 2013 1:18 AM
>Subject: Re: 2P vs 1P psf
>
>
>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:[hidden email]] On
>Behalf Of Jeff Reece
>Sent: Sunday, 17 November 2013 1:13 AM
>To: [hidden email]
>Subject: Re: 2P vs 1P psf
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>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." <[hidden email]>
>>To: [hidden email]
>>Sent: Friday, November 15, 2013 9:00 AM
>>Subject: 2P vs 1P psf
>>
>>
>>*****
>>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
>>
>>
>
>
>=

Guy Cox-2

Re: 2P vs 1P psf

In reply to this post by Jeff Reece

In terms of lateral resolution, the paper Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22 shows that once the pinhole reaches 1 Airy unit the resolution becomes the same as in widefield. As Lutz has pointed out, that isn't actually the same as saying that the PSF is identical, and axially they are very different. Check out my publication list at http://www.guycox.net/

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeff Reece
Sent: Monday, 18 November 2013 2:07 AM
To: [hidden email]
Subject: Re: 2P vs 1P psf

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

Is there a reference for the confocal psf being equal to the excitation psf when the pinhole=1AU (free access via the web is requested). It is not intuitive that the confocal psf would leave out the optics (pinhole) of the emission path.

Thanks,
Jeff

Steffen Dietzel

Re: 2P vs 1P psf

In reply to this post by Jeff Reece

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

Citable literature is sparse in that area. For two-photon and confocal
resolution formula I usually cite these two, the first one being the one
that Guy already mentioned:

G. Cox, C. J. Sheppard: Practical limits of resolution in confocal and
non-linear microscopy. In: Microscopy Research and Technique. Band 63,
Issue 1, 2004, p. 18–22, doi:10.1002/jemt.10423, PMID 14677129.

B. Amos, G. McConnell, T. Wilson: Confocal microscopy. In: E. Egelman
(Ed.): Biophysical Techniques for Characterization of Cells (=
Comprehensive Biophysics. 2). Elsevier, Academic Press, Amsterdam 2012,
ISBN 978-0-12-374920-8, 2, p. 3–23, doi:10.1016/B978-0-12-374920-8.00203-4
Author version:
http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

When it comes to three-photon excition it gets really thin. I just very
recently came accross a paper which gives the xy-resolution formula
(0.51*lambda)/(sqrt(3)*NA), see below. If anyone should have an older
citable reference. I very much would like to hear about it.

Steffen

Jung Ho Yu, Seung-Hae Kwon, Zdenˇek Petrášek3, Ok Kyu Park, Samuel
Woojoo Jun, Kwangsoo Shin, Moonkee Choi, Yong Il Park, Kyeongsoon Park,
Hyon Bin Na, Nohyun Lee, Dong Won Lee, Jeong Hyun Kim, Petra Schwille,
Taeghwan Hyeon: High-resolution three-photon biomedical imaging using
doped ZnS nanocrystals. In: Nature Materials. 12, 2013, p. 359–366,
doi:10.1038/NMAT3565

On 17.11.2013 16:06, Jeff Reece wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Is there a reference for the confocal psf being equal to the excitation psf when the pinhole=1AU (free access via the web is requested). It is not intuitive that the confocal psf would leave out the optics (pinhole) of the emission path.
>
> Thanks,
> Jeff
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27, München-Großhadern

George McNamara

Re: 2P vs 1P psf

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

3-photon confocal vs 3-photon PSF vs other modes (calculations) in
figure 2 of Schrader, Bahlmann, Hell 1997 Optik

http://www3.mpibpc.mpg.de/groups/hell/publications/pdf/Optik_104_116-124.pdf

Here is a recent paper on tri-exciton (a 3 photon process) imaging (see
references for earlier triexciton imaging):
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0064023
his study describes a simple technique that improves a recently
developed 3D sub-diffraction imaging method based on three-photon
absorption of commercially available quantum dots. The method combines
imaging of biological samples via tri-exciton generation in quantum dots
with deconvolution and spectral multiplexing, resulting in a novel
approach for multi-color imaging of even thick biological samples at a
1.4 to 1.9-fold better spatial resolution. This approach is realized on
a conventional confocal microscope equipped with standard
continuous-wave lasers. We demonstrate the potential of multi-color
tri-exciton imaging of quantum dots combined with deconvolution on viral
vesicles in lentivirally transduced cells as well as intermediate
filaments in three-dimensional clusters of mouse-derived neural stem
cells (neurospheres) and dense microtubuli arrays in myotubes formed by
stacks of differentiated C2C12 myoblasts.

enjoy,

George

On 11/18/2013 4:47 AM, Steffen Dietzel wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Citable literature is sparse in that area. For two-photon and confocal
> resolution formula I usually cite these two, the first one being the
> one that Guy already mentioned:
>
> G. Cox, C. J. Sheppard: Practical limits of resolution in confocal and
> non-linear microscopy. In: Microscopy Research and Technique. Band 63,
> Issue 1, 2004, p. 18–22, doi:10.1002/jemt.10423, PMID 14677129.
>
> B. Amos, G. McConnell, T. Wilson: Confocal microscopy. In: E. Egelman
> (Ed.): Biophysical Techniques for Characterization of Cells (=
> Comprehensive Biophysics. 2). Elsevier, Academic Press, Amsterdam
> 2012, ISBN 978-0-12-374920-8, 2, p. 3–23,
> doi:10.1016/B978-0-12-374920-8.00203-4
> Author version:
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>
>
>
> When it comes to three-photon excition it gets really thin. I just
> very recently came accross a paper which gives the xy-resolution
> formula (0.51*lambda)/(sqrt(3)*NA), see below. If anyone should have
> an older citable reference. I very much would like to hear about it.
>
> Steffen
>
> Jung Ho Yu, Seung-Hae Kwon, Zdenˇek Petrášek3, Ok Kyu Park, Samuel
> Woojoo Jun, Kwangsoo Shin, Moonkee Choi, Yong Il Park, Kyeongsoon
> Park, Hyon Bin Na, Nohyun Lee, Dong Won Lee, Jeong Hyun Kim, Petra
> Schwille, Taeghwan Hyeon: High-resolution three-photon biomedical
> imaging using doped ZnS nanocrystals. In: Nature Materials. 12, 2013,
> p. 359–366, doi:10.1038/NMAT3565
>
> On 17.11.2013 16:06, Jeff Reece wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Is there a reference for the confocal psf being equal to the
>> excitation psf when the pinhole=1AU (free access via the web is
>> requested). It is not intuitive that the confocal psf would leave
>> out the optics (pinhole) of the emission path.
>>
>> Thanks,
>> Jeff
>>
>
>

--

George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/

Jeff Reece

Re: 2P vs 1P psf

In reply to this post by Steffen Dietzel

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

I think Figure 4 in the Amos et al chapter is a great way to visualize how the excitation psf dominates the overall confocal psf for xy resolution.
Similar figures for z resolution are what we need to answer the original question, comparing confocal with 2P. So there would be Fig 4z-conf and Fig 4z-2P. Fig Fig 4z-conf would also demonstrate the utility of the pinhole in z compared to confocal xy. I cannot quickly create these myself right now but will describe them.

In both Figs 4z-conf and 4z-2P, the first row showing the graphs of the pinhole dimensions would be eliminated. In Fig 4z-conf, one might instead replace it with statements of the pinhole size in AU used for that column.

In Fig 4z-conf, the roles of excitation psf (2nd row of Fig 4) and emission psf (3rd row of Fig 4) are reversed, in that the emission psf will dominate (be smaller than) the overall psf. When the pinhole is completely closed, the emission psf will typically be much smaller than the excitation psf. As the pinhole is opened, the emission psf will gradually get as big as the excitation psf, or more accurately, as big as the standard widefield emission psf, which is usually slightly bigger than the excitation psf.

In Fig 4z-2P, it might be useful to add another row before the 2P excitation psf that shows the 1P version of the psf, at the 2P excitation wavelength, i.e. before the 2P effect occurs, so one can visualize the effect of squaring the 1P psf to get the true 2P excitation psf.
The 2P emission psf would be the WF emission psf equivalent to the open pinhole in confocal mode. The 2P excitation psf would dominate the overall 2P psf, as in the case for confocal xy.

Probably clear as mud without the real figure.

-Jeff

>________________________________
> From: Steffen Dietzel <[hidden email]>
>To: [hidden email]
>Sent: Monday, November 18, 2013 5:47 AM
>Subject: Re: 2P vs 1P psf
>
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Citable literature is sparse in that area. For two-photon and confocal
>resolution formula I usually cite these two, the first one being the one
>that Guy already mentioned:
>
>G. Cox, C. J. Sheppard: Practical limits of resolution in confocal and
>non-linear microscopy. In: Microscopy Research and Technique. Band 63,
>Issue 1, 2004, p. 18–22, doi:10.1002/jemt.10423, PMID 14677129.
>
>B. Amos, G. McConnell, T. Wilson: Confocal microscopy. In: E. Egelman
>(Ed.): Biophysical Techniques for Characterization of Cells (=
>Comprehensive Biophysics. 2). Elsevier, Academic Press, Amsterdam 2012,
>ISBN 978-0-12-374920-8, 2, p. 3–23, doi:10.1016/B978-0-12-374920-8.00203-4
>Author version:
>http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>
>
>When it comes to three-photon excition it gets really thin. I just very
>recently came accross a paper which gives the xy-resolution formula
>(0.51*lambda)/(sqrt(3)*NA), see below. If anyone should have an older
>citable reference. I very much would like to hear about it.
>
>Steffen
>
>Jung Ho Yu, Seung-Hae Kwon, Zdenˇek Petrášek3, Ok Kyu Park, Samuel
>Woojoo Jun, Kwangsoo Shin, Moonkee Choi, Yong Il Park, Kyeongsoon Park,
>Hyon Bin Na, Nohyun Lee, Dong Won Lee, Jeong Hyun Kim, Petra Schwille,
>Taeghwan Hyeon: High-resolution three-photon biomedical imaging using
>doped ZnS nanocrystals. In: Nature Materials. 12, 2013, p. 359–366,
>doi:10.1038/NMAT3565
>
>
>On 17.11.2013 16:06, Jeff Reece wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Is there a reference for the confocal psf being equal to the excitation psf when the pinhole=1AU (free access via the web is requested). It is not intuitive that the confocal psf would leave out the optics (pinhole) of the emission path.
>>
>> Thanks,
>> Jeff
>>
>
>
>--
>------------------------------------------------------------
>Steffen Dietzel, PD Dr. rer. nat
>Ludwig-Maximilians-Universität München
>Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>Head of light microscopy
>
>Mail room:
>Marchioninistr. 15, D-81377 München
>
>Building location:
>Marchioninistr. 27, München-Großhadern
>
>
>
>

Laevsky, Gary S.

Re: 2P vs 1P psf

Thank you all for the responses. I guess what I thought was simple was not the case.

As I had not done the experiments, and when I explain our setup, you’ll see our limitations, and what were some great take home points.

We cannot put a pinhole in front of our non-descanned detector (maybe we could, but it would not be trivial), and we do not have any descanned detectors on this system (it is MP only).
An additional IR block in front of the detector may be useful.
Broadband excitation of multiple flours needs to be considered. We can modify filters here.
I had no idea you approached WF psf so fast as you open a pinhole past 1 Airy.
In xy, the psf between 2P and 1P will be relatively equivalent, but not so in z.

If I am wrong on any of these points, I would gratefully like to know, off list.

Thanks so much.

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

On Nov 18, 2013, at 9:48 AM, Jeff Reece <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I think Figure 4 in the Amos et al chapter is a great way to visualize how the excitation psf dominates the overall confocal psf for xy resolution.
> Similar figures for z resolution are what we need to answer the original question, comparing confocal with 2P. So there would be Fig 4z-conf and Fig 4z-2P. Fig Fig 4z-conf would also demonstrate the utility of the pinhole in z compared to confocal xy. I cannot quickly create these myself right now but will describe them.
>
> In both Figs 4z-conf and 4z-2P, the first row showing the graphs of the pinhole dimensions would be eliminated. In Fig 4z-conf, one might instead replace it with statements of the pinhole size in AU used for that column.
>
> In Fig 4z-conf, the roles of excitation psf (2nd row of Fig 4) and emission psf (3rd row of Fig 4) are reversed, in that the emission psf will dominate (be smaller than) the overall psf. When the pinhole is completely closed, the emission psf will typically be much smaller than the excitation psf. As the pinhole is opened, the emission psf will gradually get as big as the excitation psf, or more accurately, as big as the standard widefield emission psf, which is usually slightly bigger than the excitation psf.
>
> In Fig 4z-2P, it might be useful to add another row before the 2P excitation psf that shows the 1P version of the psf, at the 2P excitation wavelength, i.e. before the 2P effect occurs, so one can visualize the effect of squaring the 1P psf to get the true 2P excitation psf.
> The 2P emission psf would be the WF emission psf equivalent to the open pinhole in confocal mode. The 2P excitation psf would dominate the overall 2P psf, as in the case for confocal xy.
>
> Probably clear as mud without the real figure.
>
> -Jeff
>
>
>
>
>> ________________________________
>> From: Steffen Dietzel <[hidden email]>
>> To: [hidden email]
>> Sent: Monday, November 18, 2013 5:47 AM
>> Subject: Re: 2P vs 1P psf
>>
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Citable literature is sparse in that area. For two-photon and confocal
>> resolution formula I usually cite these two, the first one being the one
>> that Guy already mentioned:
>>
>> G. Cox, C. J. Sheppard: Practical limits of resolution in confocal and
>> non-linear microscopy. In: Microscopy Research and Technique. Band 63,
>> Issue 1, 2004, p. 18–22, doi:10.1002/jemt.10423, PMID 14677129.
>>
>> B. Amos, G. McConnell, T. Wilson: Confocal microscopy. In: E. Egelman
>> (Ed.): Biophysical Techniques for Characterization of Cells (=
>> Comprehensive Biophysics. 2). Elsevier, Academic Press, Amsterdam 2012,
>> ISBN 978-0-12-374920-8, 2, p. 3–23, doi:10.1016/B978-0-12-374920-8.00203-4
>> Author version:
>> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>>
>>
>> When it comes to three-photon excition it gets really thin. I just very
>> recently came accross a paper which gives the xy-resolution formula
>> (0.51*lambda)/(sqrt(3)*NA), see below. If anyone should have an older
>> citable reference. I very much would like to hear about it.
>>
>> Steffen
>>
>> Jung Ho Yu, Seung-Hae Kwon, Zdenˇek Petrášek3, Ok Kyu Park, Samuel
>> Woojoo Jun, Kwangsoo Shin, Moonkee Choi, Yong Il Park, Kyeongsoon Park,
>> Hyon Bin Na, Nohyun Lee, Dong Won Lee, Jeong Hyun Kim, Petra Schwille,
>> Taeghwan Hyeon: High-resolution three-photon biomedical imaging using
>> doped ZnS nanocrystals. In: Nature Materials. 12, 2013, p. 359–366,
>> doi:10.1038/NMAT3565
>>
>>
>> On 17.11.2013 16:06, Jeff Reece wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Is there a reference for the confocal psf being equal to the excitation psf when the pinhole=1AU (free access via the web is requested). It is not intuitive that the confocal psf would leave out the optics (pinhole) of the emission path.
>>>
>>> Thanks,
>>> Jeff
>>>
>>
>>
>> --
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München
>> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>> Head of light microscopy
>>
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>>
>> Building location:
>> Marchioninistr. 27, München-Großhadern
>>
>>
>>
>>

Steffen Dietzel

Re: 2P vs 1P psf

In reply to this post by George McNamara

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

George,

thanks, I knew Anje Sporber's Paper but not the other one. Anyway, they
are good examples of what I meant: They show improved resolution but do
not give a FWHM formula a plain biologist can make use of. Again, if
somebody has an older reference with the formula for 3-photon
excitation, I would appreciate being pointed to it.

Steffen

On 18.11.2013 14:49, George McNamara wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> 3-photon confocal vs 3-photon PSF vs other modes (calculations) in
> figure 2 of Schrader, Bahlmann, Hell 1997 Optik
>
> http://www3.mpibpc.mpg.de/groups/hell/publications/pdf/Optik_104_116-124.pdf
>
>
> Here is a recent paper on tri-exciton (a 3 photon process) imaging (see
> references for earlier triexciton imaging):
> http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0064023

>
>
>
> enjoy,
>
> George
>
> On 11/18/2013 4:47 AM, Steffen Dietzel wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Citable literature is sparse in that area. For two-photon and confocal
>> resolution formula I usually cite these two, the first one being the
>> one that Guy already mentioned:
>>
>> G. Cox, C. J. Sheppard: Practical limits of resolution in confocal and
>> non-linear microscopy. In: Microscopy Research and Technique. Band 63,
>> Issue 1, 2004, p. 18–22, doi:10.1002/jemt.10423, PMID 14677129.
>>
>> B. Amos, G. McConnell, T. Wilson: Confocal microscopy. In: E. Egelman
>> (Ed.): Biophysical Techniques for Characterization of Cells (=
>> Comprehensive Biophysics. 2). Elsevier, Academic Press, Amsterdam
>> 2012, ISBN 978-0-12-374920-8, 2, p. 3–23,
>> doi:10.1016/B978-0-12-374920-8.00203-4
>> Author version:
>> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>>
>>
>>
>> When it comes to three-photon excition it gets really thin. I just
>> very recently came accross a paper which gives the xy-resolution
>> formula (0.51*lambda)/(sqrt(3)*NA), see below. If anyone should have
>> an older citable reference. I very much would like to hear about it.
>>
>> Steffen
>>
>> Jung Ho Yu, Seung-Hae Kwon, Zdenˇek Petrášek3, Ok Kyu Park, Samuel
>> Woojoo Jun, Kwangsoo Shin, Moonkee Choi, Yong Il Park, Kyeongsoon
>> Park, Hyon Bin Na, Nohyun Lee, Dong Won Lee, Jeong Hyun Kim, Petra
>> Schwille, Taeghwan Hyeon: High-resolution three-photon biomedical
>> imaging using doped ZnS nanocrystals. In: Nature Materials. 12, 2013,
>> p. 359–366, doi:10.1038/NMAT3565
>>
>> On 17.11.2013 16:06, Jeff Reece wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Is there a reference for the confocal psf being equal to the
>>> excitation psf when the pinhole=1AU (free access via the web is
>>> requested). It is not intuitive that the confocal psf would leave out
>>> the optics (pinhole) of the emission path.
>>>
>>> Thanks,
>>> Jeff
>>>
>>
>>
>
>