pinhole and voxel

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear All,

I'm quite new in the confocal world, I'll start a PhD and I will have to use confocal.
I well understand the principle of pinhole and I can figure out its effect on XY resolution but I can't figure out precisely what effect it have about Z resolution.
If the pinhole leads to have 1 AU, what will be the z resolution ? How to calculate it ?
Knowing the pinhole size we can calculte XY resolution by calculated the backprojected pinhole radius but how this value is related to z resolution ?

Thanks for help

Max

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[Boswell, Carl A - (cboswell)]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Max,
In general, what you get with the pinhole at 1 Airy unit is only the light
from the sample that is in focus.  What is in focus (also called "depth of
field") depends on the NA of the objective.  You can find a calculator at:
http://www.mcb.arizona.edu/ipc/dv/depth_of_field.htm that will give you an
idea of the relationship of NA and depth of field.  As you can see by the
calculator, and by lateral resolution page:
http://www.mcb.arizona.edu/ipc/dv/zoom_Page.htm, the z resolution is
inherently lower (worse) than the lateral resolution.  Other mathmatical
approaches will calculate somewhat different values, but the absolute values
are similar and the relationships are the same.

Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "[hidden email]" <[hidden email]>
To: <[hidden email]>
Sent: Tuesday, October 09, 2007 2:56 PM
Subject: pinhole and voxel


Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear All,

I'm quite new in the confocal world, I'll start a PhD and I will have to use
confocal.
I well understand the principle of pinhole and I can figure out its effect
on XY resolution but I can't figure out precisely what effect it have about
Z resolution.
If the pinhole leads to have 1 AU, what will be the z resolution ? How to
calculate it ?
Knowing the pinhole size we can calculte XY resolution by calculated the
backprojected pinhole radius but how this value is related to z resolution ?

Thanks for help

Max

------------------------ ALICE C'EST ENCORE MIEUX AVEC CANAL+ LE BOUQUET
! ---------------
Découvrez vite l'offre exclusive ALICEBOX et CANAL+ LE BOUQUET, en cliquant
ici http://alicebox.fr
Soumis à conditions.

[Kevin Braeckmans]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Max,

Maybe some of the more knowledgeable scientists on this list may give you a
more detailed answer, but as far as I understand it is the same back
projection of the pinhole that you need to calculate the axial resolution.

If you know the 3D PSF of the objective lens, you can calculate the
backprojection of the pinhole in 3D, which is the convolution product of the
pinhole with the PSF (which is the PSF itself for an infinitely small
pinhole). The total confocal resolution can then be calculated as the
product of this backprojected pinhole distribution and the objective's PSF.

There is a very comprehensive chapter on the fundamentals of resolution of
different types of microscopes (confocal, widefield and Multi-photon) in
Alberto Diaspro's excellent handbook:

J. E. N. Jonkman and E. H. K. Stelzer. Resolution and contrast in confocal
and two-photon microscopy.
In A. Diaspro, editor, Confocal and Two-Photon Microscopy: Foundations,
Applications and Advances, book chapter 5, pages 101–125. Wiley-Liss, Inc.,
New York, USA, 2002.


I really recommend reading this chapter. It is very clearly written and also
explains the difference between optical sectioning and axial resolution.

Best regards,

Kevin


Kevin Braeckmans, Ph.D.
Lab. General Biochemistry & Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: [phone]
Fax: [phone]
E-mail: [hidden email]

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens [hidden email]
> Verzonden: dinsdag 9 oktober 2007 23:57
> Aan: [hidden email]
> Onderwerp: pinhole and voxel
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear All,
>
> I'm quite new in the confocal world, I'll start a PhD and I will have
> to use confocal.
> I well understand the principle of pinhole and I can figure out its
> effect on XY resolution but I can't figure out precisely what effect it
> have about Z resolution.
> If the pinhole leads to have 1 AU, what will be the z resolution ? How
> to calculate it ?
> Knowing the pinhole size we can calculte XY resolution by calculated
> the backprojected pinhole radius but how this value is related to z
> resolution ?
>
> Thanks for help
>
> Max
>
> ------------------------ ALICE C'EST ENCORE MIEUX AVEC CANAL+ LE
> BOUQUET ! ---------------
> Découvrez vite l'offre exclusive ALICEBOX et CANAL+ LE BOUQUET, en
> cliquant ici http://alicebox.fr
> Soumis à conditions.

[Knecht, David]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

The equations being used here don't take into account the size of the  
excitation and emission pinholes.  This can vary at the emission end  
for an LSCM.  At the excitation end I don't know if all the  
instruments are the same.  For the spinning disks, how do you compare  
the resolution/depth of field of a nipkow spinning disk, with a CARV/
Leica type device which have different pinhole sizes for excitation  
and emission? Am I correct that the laser vs. non-laser of the two  
spinning disks makes no difference?  Dave

Dr. David Knecht
Department of Molecular and Cell Biology
Co-head Flow Cytometry and Confocal Microscopy Facility
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)


On Oct 10, 2007, at 3:47 AM, Kevin Braeckmans wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear Max,
>
> Maybe some of the more knowledgeable scientists on this list may  
> give you a
> more detailed answer, but as far as I understand it is the same back
> projection of the pinhole that you need to calculate the axial  
> resolution.
>
> If you know the 3D PSF of the objective lens, you can calculate the
> backprojection of the pinhole in 3D, which is the convolution  
> product of the
> pinhole with the PSF (which is the PSF itself for an infinitely small
> pinhole). The total confocal resolution can then be calculated as the
> product of this backprojected pinhole distribution and the  
> objective's PSF.
>
> There is a very comprehensive chapter on the fundamentals of  
> resolution of
> different types of microscopes (confocal, widefield and Multi-
> photon) in
> Alberto Diaspro's excellent handbook:
>
> J. E. N. Jonkman and E. H. K. Stelzer. Resolution and contrast in  
> confocal
> and two-photon microscopy.
> In A. Diaspro, editor, Confocal and Two-Photon Microscopy:  
> Foundations,
> Applications and Advances, book chapter 5, pages 101–125. Wiley-
> Liss, Inc.,
> New York, USA, 2002.
>
>
> I really recommend reading this chapter. It is very clearly written  
> and also
> explains the difference between optical sectioning and axial  
> resolution.
>
> Best regards,
>
> Kevin
>
>
> Kevin Braeckmans, Ph.D.
> Lab. General Biochemistry & Physical Pharmacy
> Ghent University
> Harelbekestraat 72
> 9000 Ghent
> Belgium
> Tel: [phone]
> Fax: [phone]
> E-mail: [hidden email]
>
>
>> -----Oorspronkelijk bericht-----
>> Van: Confocal Microscopy List [mailto:[hidden email]]
>> Namens [hidden email]
>> Verzonden: dinsdag 9 oktober 2007 23:57
>> Aan: [hidden email]
>> Onderwerp: pinhole and voxel
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Dear All,
>>
>> I'm quite new in the confocal world, I'll start a PhD and I will have
>> to use confocal.
>> I well understand the principle of pinhole and I can figure out its
>> effect on XY resolution but I can't figure out precisely what  
>> effect it
>> have about Z resolution.
>> If the pinhole leads to have 1 AU, what will be the z resolution ?  
>> How
>> to calculate it ?
>> Knowing the pinhole size we can calculte XY resolution by calculated
>> the backprojected pinhole radius but how this value is related to z
>> resolution ?
>>
>> Thanks for help
>>
>> Max
>>
>> ------------------------ ALICE C'EST ENCORE MIEUX AVEC CANAL+ LE
>> BOUQUET ! ---------------
>> Découvrez vite l'offre exclusive ALICEBOX et CANAL+ LE BOUQUET, en
>> cliquant ici http://alicebox.fr
>> Soumis à conditions.

[Kevin Braeckmans]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

The equations I was thinking about in this book chapter are the ones in
paragraphs 2.3 and 3.3. No a priori assumption is made there about pinhole
sizes. They rather give the general formalism how to calculate effective
PSFs and the optical sectioning capability. I found this to be very
instructive. As I said in my previous message, the finite pinhole size can
be taken into account by calculating the convolution product of the pinhole
with the objective's PSF. This would be the effective PSF at the detection
side (h_det in the book chapter).

Sorry, don't know about the calculations for a spinning disk system.

Best regards,

Kevin



Kevin Braeckmans, Ph.D.
Lab. General Biochemistry and Physical Pharmacy
Ghent University
Harelbekestraat 72
9000 Ghent
Belgium
Tel: [phone]
Fax: [phone]

> -----Oorspronkelijk bericht-----
> Van: Confocal Microscopy List [mailto:[hidden email]]
> Namens David Knecht-charter
> Verzonden: donderdag 11 oktober 2007 3:27
> Aan: [hidden email]
> Onderwerp: Re: pinhole and voxel
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> The equations being used here don't take into account the size of the
> excitation and emission pinholes.  This can vary at the emission end
> for an LSCM.  At the excitation end I don't know if all the
> instruments are the same.  For the spinning disks, how do you compare
> the resolution/depth of field of a nipkow spinning disk, with a CARV/
> Leica type device which have different pinhole sizes for excitation
> and emission? Am I correct that the laser vs. non-laser of the two
> spinning disks makes no difference?  Dave
>
> Dr. David Knecht
> Department of Molecular and Cell Biology
> Co-head Flow Cytometry and Confocal Microscopy Facility
> U-3125
> 91 N. Eagleville Rd.
> University of Connecticut
> Storrs, CT 06269
> 860-486-2200
> 860-486-4331 (fax)
>
>
> On Oct 10, 2007, at 3:47 AM, Kevin Braeckmans wrote:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Dear Max,
> >
> > Maybe some of the more knowledgeable scientists on this list may
> > give you a
> > more detailed answer, but as far as I understand it is the same back
> > projection of the pinhole that you need to calculate the axial
> > resolution.
> >
> > If you know the 3D PSF of the objective lens, you can calculate the
> > backprojection of the pinhole in 3D, which is the convolution
> > product of the
> > pinhole with the PSF (which is the PSF itself for an infinitely small
> > pinhole). The total confocal resolution can then be calculated as the
> > product of this backprojected pinhole distribution and the
> > objective's PSF.
> >
> > There is a very comprehensive chapter on the fundamentals of
> > resolution of
> > different types of microscopes (confocal, widefield and Multi-
> > photon) in
> > Alberto Diaspro's excellent handbook:
> >
> > J. E. N. Jonkman and E. H. K. Stelzer. Resolution and contrast in
> > confocal
> > and two-photon microscopy.
> > In A. Diaspro, editor, Confocal and Two-Photon Microscopy:
> > Foundations,
> > Applications and Advances, book chapter 5, pages 101–125. Wiley-
> > Liss, Inc.,
> > New York, USA, 2002.
> >
> >
> > I really recommend reading this chapter. It is very clearly written
> > and also
> > explains the difference between optical sectioning and axial
> > resolution.
> >
> > Best regards,
> >
> > Kevin
> >
> >
> > Kevin Braeckmans, Ph.D.
> > Lab. General Biochemistry & Physical Pharmacy
> > Ghent University
> > Harelbekestraat 72
> > 9000 Ghent
> > Belgium
> > Tel: [phone]
> > Fax: [phone]
> > E-mail: [hidden email]
> >
> >
> >> -----Oorspronkelijk bericht-----
> >> Van: Confocal Microscopy List [mailto:[hidden email]]
> >> Namens [hidden email]
> >> Verzonden: dinsdag 9 oktober 2007 23:57
> >> Aan: [hidden email]
> >> Onderwerp: pinhole and voxel
> >>
> >> Search the CONFOCAL archive at
> >> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >>
> >> Dear All,
> >>
> >> I'm quite new in the confocal world, I'll start a PhD and I will
> have
> >> to use confocal.
> >> I well understand the principle of pinhole and I can figure out its
> >> effect on XY resolution but I can't figure out precisely what
> >> effect it
> >> have about Z resolution.
> >> If the pinhole leads to have 1 AU, what will be the z resolution ?
> >> How
> >> to calculate it ?
> >> Knowing the pinhole size we can calculte XY resolution by calculated
> >> the backprojected pinhole radius but how this value is related to z
> >> resolution ?
> >>
> >> Thanks for help
> >>
> >> Max
> >>
> >> ------------------------ ALICE C'EST ENCORE MIEUX AVEC CANAL+ LE
> >> BOUQUET ! ---------------
> >> Découvrez vite l'offre exclusive ALICEBOX et CANAL+ LE BOUQUET, en
> >> cliquant ici http://alicebox.fr
> >> Soumis à conditions.