Estimating field of view in an LSM

[Craig Brideau]

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

I was wondering how I estimate the field of view for a given lens in a
laser scanning microscope?  I know it will depend on the entrance
angle of the beam into the back aperture of the lens, but I don't know
how to translate this into lateral focal point displacement for a
given working distance/NA/magnification of lens.  Can anyone point me
towards some good references?

Thanks,

Craig

[Craig Brideau]

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

Just to clarify, I am doing multiphoton with a single PMT, so I'm
trying to measure the field that the laser is focused over at the
focal plane of the objective for a given input beam deflection.

Craig

On 9/20/07, Craig Brideau <[hidden email]> wrote:

> I was wondering how I estimate the field of view for a given lens in a
> laser scanning microscope?  I know it will depend on the entrance
> angle of the beam into the back aperture of the lens, but I don't know
> how to translate this into lateral focal point displacement for a
> given working distance/NA/magnification of lens.  Can anyone point me
> towards some good references?
>
> Thanks,
>
> Craig
>

[Steffen Dietzel]

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

Craig,

for the theory I don't know, but to actually meassure the field of
view (or the size of your pixels) you can use a 1951 USAF Resolution
Test Chart. See e.g.
http://en.wikipedia.org/wiki/1951_USAF_Resolution_Test_Chart

There are several suppliers you can google for. I got mine from
Edmund Optics. It has a mirror coating on the top surface so that it
can be used in reflection mode.

Steffen



At 22:16 20.09.2007, you wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Just to clarify, I am doing multiphoton with a single PMT, so I'm
>trying to measure the field that the laser is focused over at the
>focal plane of the objective for a given input beam deflection.
>
>Craig
>
>On 9/20/07, Craig Brideau <[hidden email]> wrote:
> > I was wondering how I estimate the field of view for a given lens in a
> > laser scanning microscope?  I know it will depend on the entrance
> > angle of the beam into the back aperture of the lens, but I don't know
> > how to translate this into lateral focal point displacement for a
> > given working distance/NA/magnification of lens.  Can anyone point me
> > towards some good references?
> >
> > Thanks,
> >
> > Craig
> >

[Craig Brideau]

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

Yeah, I want to be able to predict the field ahead of time BEFORE I
buy an objective.  I'll have to keep looking in to the theory.  If it
was a simple lens I could just use some ray trace equations, but
microscope objectives are very much 'black box' devices due to their
complexity, and proprietary design.  Still, good advice for
characterizing my existing lenses.

Thanks,

Craig

On 9/21/07, Steffen Dietzel <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Craig,
>
> for the theory I don't know, but to actually meassure the field of
> view (or the size of your pixels) you can use a 1951 USAF Resolution
> Test Chart. See e.g.
> http://en.wikipedia.org/wiki/1951_USAF_Resolution_Test_Chart
>
> There are several suppliers you can google for. I got mine from
> Edmund Optics. It has a mirror coating on the top surface so that it
> can be used in reflection mode.
>
> Steffen
>
>
>
> At 22:16 20.09.2007, you wrote:
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Just to clarify, I am doing multiphoton with a single PMT, so I'm
> >trying to measure the field that the laser is focused over at the
> >focal plane of the objective for a given input beam deflection.
> >
> >Craig
> >
> >On 9/20/07, Craig Brideau <[hidden email]> wrote:
> > > I was wondering how I estimate the field of view for a given lens in a
> > > laser scanning microscope?  I know it will depend on the entrance
> > > angle of the beam into the back aperture of the lens, but I don't know
> > > how to translate this into lateral focal point displacement for a
> > > given working distance/NA/magnification of lens.  Can anyone point me
> > > towards some good references?
> > >
> > > Thanks,
> > >
> > > Craig
> > >
>

[Julio Vazquez]

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

-

Hi Craig, 

I'm venturing into long-faded and possibly confused memories here, but I'm inclined to think that you can determine this from geometrical optics. Basically, a parallel bundle of rays that enters the lens at a perpendicular (normal) angle will be focused to the focal spot (center of the field of view), while a parallel bundle of rays that enters the lens at an angle alpha along one of the axes (alpha being equal to the half-cone of acceptance, basically the angle used to measure NA) will be focused at a distance from the center of the field such that d/f = tan(alpha) .  You can verify this by a simple ray diagram.  For any arbitrary angle you would have to consider the deviation along both axes to know exactly where the beam will be focused. I don't know if there are  additional subtleties when more realistic "thick lenses", "compound lenses" or infinity lenses are involved...

Actually, I just consulted the Confocal Handbook, 3rd Edition, to make sure I was not talking nonsense, and Chapter 9 contains a diagram and gives some formulas that seem to confirm the above statement... You may want to check it out, specifically pp 208-209.

 I would assume that in practice the actual field of view will be limited by the dimensions of the laser beam and objective, and the size of the scanning mirror, since at some point, you can't increase the angle without losing power, getting some "vignetting-like" effect, or getting geometrical distortions...

Just for reference, on our Zeiss LSM 510, the field dimensions with a 20x lens at zoom 1 are 460.7 x 460.7 microns, and they would be half that much with a 40x or with a 20x at zoom 2 (independently of NA). On our Leica SP, the values are 500x500 microns for 20x at zoom 1, etc...  

--

Julio Vazquez, 

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

[hidden email]

http://www.fhcrc.org/

On Sep 21, 2007, at 1:29 PM, Craig Brideau wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Yeah, I want to be able to predict the field ahead of time BEFORE I

buy an objective.  I'll have to keep looking in to the theory.  If it

was a simple lens I could just use some ray trace equations, but

microscope objectives are very much 'black box' devices due to their

complexity, and proprietary design.  Still, good advice for

characterizing my existing lenses.

Thanks,

Craig

On 9/21/07, Steffen Dietzel <[hidden email]> wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Craig,

for the theory I don't know, but to actually meassure the field of

view (or the size of your pixels) you can use a 1951 USAF Resolution

Test Chart. See e.g.

http://en.wikipedia.org/wiki/1951_USAF_Resolution_Test_Chart

There are several suppliers you can google for. I got mine from

Edmund Optics. It has a mirror coating on the top surface so that it

can be used in reflection mode.

Steffen

At 22:16 20.09.2007, you wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Just to clarify, I am doing multiphoton with a single PMT, so I'm

trying to measure the field that the laser is focused over at the

focal plane of the objective for a given input beam deflection.

Craig

On 9/20/07, Craig Brideau <[hidden email]> wrote:

I was wondering how I estimate the field of view for a given lens in a

laser scanning microscope?  I know it will depend on the entrance

angle of the beam into the back aperture of the lens, but I don't know

how to translate this into lateral focal point displacement for a

given working distance/NA/magnification of lens.  Can anyone point me

towards some good references?

Thanks,

Craig

[Guy Cox]

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

I'm not sure I see the problem.  Surely
you can find or borrow one lens at least -
the rest is just scaling.  Rather than buy
an expensive test sample I'd suggest
bleaching an area on a disposable sample
and measuring the bleached area.  Or for
a low-power objective mark some lines with
a fine black pen on one of the Chroma
fluorescent plastic slides.  

If you really want to do it by ray-tracing
I suggest not doing it through the objective
(since, as you say, it's not quite clear where
the bfp of the objective is) but transferring
your angles to displacements at the primary image
plane - you can rely on the objective to do a
faithful demagnification of the image plane to
the sample - of course this does asssume you are
forming your image at the correct plane.  

                                   Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
Phone [phone]     Fax [phone]
Mobile [phone]
______________________________________________
     http://www.guycox.net
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
Sent: Saturday, 22 September 2007 6:30 AM
To: [hidden email]
Subject: Re: Estimating field of view in an LSM

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

Yeah, I want to be able to predict the field ahead of time BEFORE I buy an objective.  I'll have to keep looking in to the theory.  If it was a simple lens I could just use some ray trace equations, but microscope objectives are very much 'black box' devices due to their complexity, and proprietary design.  Still, good advice for characterizing my existing lenses.

Thanks,

Craig

On 9/21/07, Steffen Dietzel <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Craig,
>
> for the theory I don't know, but to actually meassure the field of
> view (or the size of your pixels) you can use a 1951 USAF Resolution
> Test Chart. See e.g.
> http://en.wikipedia.org/wiki/1951_USAF_Resolution_Test_Chart
>
> There are several suppliers you can google for. I got mine from Edmund
> Optics. It has a mirror coating on the top surface so that it can be
> used in reflection mode.
>
> Steffen
>
>
>
> At 22:16 20.09.2007, you wrote:
> >Search the CONFOCAL archive at
> >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> >Just to clarify, I am doing multiphoton with a single PMT, so I'm
> >trying to measure the field that the laser is focused over at the
> >focal plane of the objective for a given input beam deflection.
> >
> >Craig
> >
> >On 9/20/07, Craig Brideau <[hidden email]> wrote:
> > > I was wondering how I estimate the field of view for a given lens
> > > in a laser scanning microscope?  I know it will depend on the
> > > entrance angle of the beam into the back aperture of the lens, but
> > > I don't know how to translate this into lateral focal point
> > > displacement for a given working distance/NA/magnification of
> > > lens.  Can anyone point me towards some good references?
> > >
> > > Thanks,
> > >
> > > Craig
> > >
>

Internal Virus Database is out-of-date.
Checked by AVG Free Edition.
Version: 7.5.487 / Virus Database: 269.13.19/1008 - Release Date: 14/09/2007 8:59 AM
 

Internal Virus Database is out-of-date.
Checked by AVG Free Edition.
Version: 7.5.487 / Virus Database: 269.13.19/1008 - Release Date: 14/09/2007 8:59 AM
 

[Craig Brideau]

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

This seems to make sense for an approximate calculation.  I'll sit
down and run the numbers and see if it agrees with some known
situations.

Thanks!

Craig

On 9/21/07, Julio Vazquez <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> -Hi Craig,
>
> I'm venturing into long-faded and possibly confused memories here, but I'm
> inclined to think that you can determine this from geometrical optics.
> Basically, a parallel bundle of rays that enters the lens at a perpendicular
> (normal) angle will be focused to the focal spot (center of the field of
> view), while a parallel bundle of rays that enters the lens at an angle
> alpha along one of the axes (alpha being equal to the half-cone of
> acceptance, basically the angle used to measure NA) will be focused at a
> distance from the center of the field such that d/f = tan(alpha) .  You can
> verify this by a simple ray diagram.  For any arbitrary angle you would have
> to consider the deviation along both axes to know exactly where the beam
> will be focused. I don't know if there are  additional subtleties when more
> realistic "thick lenses", "compound lenses" or infinity lenses are
> involved...
>
> Actually, I just consulted the Confocal Handbook, 3rd Edition, to make sure
> I was not talking nonsense, and Chapter 9 contains a diagram and gives some
> formulas that seem to confirm the above statement... You may want to check
> it out, specifically pp 208-209.
>
>  I would assume that in practice the actual field of view will be limited by
> the dimensions of the laser beam and objective, and the size of the scanning
> mirror, since at some point, you can't increase the angle without losing
> power, getting some "vignetting-like" effect, or getting geometrical
> distortions...
>
> Just for reference, on our Zeiss LSM 510, the field dimensions with a 20x
> lens at zoom 1 are 460.7 x 460.7 microns, and they would be half that much
> with a 40x or with a 20x at zoom 2 (independently of NA). On our Leica SP,
> the values are 500x500 microns for 20x at zoom 1, etc...
>
>
> --
> Julio Vazquez,
> Fred Hutchinson Cancer Research Center
> 1100 Fairview Ave. N.,  mailstop DE-512
> Seattle, WA 98109-1024
>
>
> [hidden email]
> http://www.fhcrc.org/
>
>
>
>
>
> On Sep 21, 2007, at 1:29 PM, Craig Brideau wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Yeah, I want to be able to predict the field ahead of time BEFORE I
> buy an objective.  I'll have to keep looking in to the theory.  If it
> was a simple lens I could just use some ray trace equations, but
> microscope objectives are very much 'black box' devices due to their
> complexity, and proprietary design.  Still, good advice for
> characterizing my existing lenses.
>
> Thanks,
>
> Craig
>
> On 9/21/07, Steffen Dietzel <[hidden email]> wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Craig,
>
> for the theory I don't know, but to actually meassure the field of
> view (or the size of your pixels) you can use a 1951 USAF Resolution
> Test Chart. See e.g.
> http://en.wikipedia.org/wiki/1951_USAF_Resolution_Test_Chart
>
> There are several suppliers you can google for. I got mine from
> Edmund Optics. It has a mirror coating on the top surface so that it
> can be used in reflection mode.
>
> Steffen
>
>
>
> At 22:16 20.09.2007, you wrote:
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Just to clarify, I am doing multiphoton with a single PMT, so I'm
> trying to measure the field that the laser is focused over at the
> focal plane of the objective for a given input beam deflection.
>
> Craig
>
> On 9/20/07, Craig Brideau <[hidden email]> wrote:
> I was wondering how I estimate the field of view for a given lens in a
> laser scanning microscope?  I know it will depend on the entrance
> angle of the beam into the back aperture of the lens, but I don't know
> how to translate this into lateral focal point displacement for a
> given working distance/NA/magnification of lens.  Can anyone point me
> towards some good references?
>
> Thanks,
>
> Craig
>
>
>
>
>

[Craig Brideau]

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

Also a very good point.  Transferring from the image plane is
straightforward.  Again, I'll try a couple methods and compare them to
known situations.

Craig


On 9/22/07, Guy Cox <[hidden email]> wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I'm not sure I see the problem.  Surely
> you can find or borrow one lens at least -
> the rest is just scaling.  Rather than buy
> an expensive test sample I'd suggest
> bleaching an area on a disposable sample
> and measuring the bleached area.  Or for
> a low-power objective mark some lines with
> a fine black pen on one of the Chroma
> fluorescent plastic slides.
>
> If you really want to do it by ray-tracing
> I suggest not doing it through the objective
> (since, as you say, it's not quite clear where
> the bfp of the objective is) but transferring
> your angles to displacements at the primary image
> plane - you can rely on the objective to do a
> faithful demagnification of the image plane to
> the sample - of course this does asssume you are
> forming your image at the correct plane.
>
>                                    Guy
>
>
>
> Optical Imaging Techniques in Cell Biology
> by Guy Cox    CRC Press / Taylor & Francis
>     http://www.guycox.com/optical.htm
> ______________________________________________
> Associate Professor Guy Cox, MA, DPhil(Oxon)
> Electron Microscope Unit, Madsen Building F09,
> University of Sydney, NSW 2006
> ______________________________________________
> Phone [phone]     Fax [phone]
> Mobile [phone]
> ______________________________________________
>      http://www.guycox.net
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Craig Brideau
> Sent: Saturday, 22 September 2007 6:30 AM
> To: [hidden email]
> Subject: Re: Estimating field of view in an LSM
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Yeah, I want to be able to predict the field ahead of time BEFORE I buy an objective.  I'll have to keep looking in to the theory.  If it was a simple lens I could just use some ray trace equations, but microscope objectives are very much 'black box' devices due to their complexity, and proprietary design.  Still, good advice for characterizing my existing lenses.
>
> Thanks,
>
> Craig
>
> On 9/21/07, Steffen Dietzel <[hidden email]> wrote:
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Craig,
> >
> > for the theory I don't know, but to actually meassure the field of
> > view (or the size of your pixels) you can use a 1951 USAF Resolution
> > Test Chart. See e.g.
> > http://en.wikipedia.org/wiki/1951_USAF_Resolution_Test_Chart
> >
> > There are several suppliers you can google for. I got mine from Edmund
> > Optics. It has a mirror coating on the top surface so that it can be
> > used in reflection mode.
> >
> > Steffen
> >
> >
> >
> > At 22:16 20.09.2007, you wrote:
> > >Search the CONFOCAL archive at
> > >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> > >
> > >Just to clarify, I am doing multiphoton with a single PMT, so I'm
> > >trying to measure the field that the laser is focused over at the
> > >focal plane of the objective for a given input beam deflection.
> > >
> > >Craig
> > >
> > >On 9/20/07, Craig Brideau <[hidden email]> wrote:
> > > > I was wondering how I estimate the field of view for a given lens
> > > > in a laser scanning microscope?  I know it will depend on the
> > > > entrance angle of the beam into the back aperture of the lens, but
> > > > I don't know how to translate this into lateral focal point
> > > > displacement for a given working distance/NA/magnification of
> > > > lens.  Can anyone point me towards some good references?
> > > >
> > > > Thanks,
> > > >
> > > > Craig
> > > >
> >
>
> Internal Virus Database is out-of-date.
> Checked by AVG Free Edition.
> Version: 7.5.487 / Virus Database: 269.13.19/1008 - Release Date: 14/09/2007 8:59 AM
>
>
> Internal Virus Database is out-of-date.
> Checked by AVG Free Edition.
> Version: 7.5.487 / Virus Database: 269.13.19/1008 - Release Date: 14/09/2007 8:59 AM
>
>

[Barbara Foster]

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

This is one of the easiest and most useful of calculations:

The full field of view of an objective is dependent on two factors:  the magnification of the objective and the FIELD NUMBER of the eyepiece.  The field number of the eyepiece is determined by the distance across a baffle which is about about 2 cm up, inside the bottom of the eyepiece. However, you don't need to measure it:  the value, in mm, is engraved on the eyepiece. Older or simpler microscopes typically have field numbers on the order of 18mm.  Newer microscopes and research stands typically range from 22mm to 25 mm; ultra wide eyepieces may go as high as about 35mm.

Please note that I said "full field of view" because the field aperture iris (located at the light port in the base of the microscope can be used to decrease the size of the field of view. This control is valuable if you have highly scattering features:  simply move the object of interest to the center of the field and close the image of the field iris around it. For most samples, you will be surprised at how much clearer and crisper the image becomes... all without any loss of resolution. 

As for the calculation:  Field of view = field number/magnification of the objective*

Note the asterisk.  The if there is any other INTERMEDIATE magnifier, multiply the Mobj x Mintermediate.  The eyepiece is NOT part of this equation. 

On a more empirical note, you can actually measure the REAL diameter of the field of view directly using a stage micrometer.  This approach works fairly well for everything except the very lowest magnifications.  In that instance, you need some reference point in the eyepiece (cross hair or an eyepiece micrometer).  Align the stage micrometer to one side of the field and take a reading to the reference point then align the stage micrometer to the other side of the field and measure to the reference point.  Simply add the two values together.  This approach is especially useful with cameras because the addition of the phototube and the camera chip frequently reduce the field of view seen through the microscope. 

Hope this was helpful.

Best regards
Barbara Foster, President

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At 03:00 PM 9/23/2007, Craig Brideau wrote:

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

I was wondering how I estimate the field of view for a given lens in a
laser scanning microscope?  I know it will depend on the entrance
angle of the beam into the back aperture of the lens, but I don't know
how to translate this into lateral focal point displacement for a
given working distance/NA/magnification of lens.  Can anyone point me
towards some good references?

Thanks,

Craig