Objective back focal plane vs. exit pupil plane locations

[Kyle Michael Douglass]

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Hi all,
I'm making an illustration of a microscope objective to demonstrate important concepts in through-the-objective TIRF illumination and ran into a question that I haven't been able to find an answer for.

For a standard, infinity-corrected objective, are there any rules for determining the location of the exit pupil relative to the back focal plane? Is it manufacturer-dependent? I know that the two planes are not coplanar for any general optical system, but in my experience with 100X, NA >= 1.4 oil objectives the two planes are pretty close to one another, lying somewhere near the back edge of the objective barrel.

Would it be "incorrect" to illustrate them as coplanar? I'd greatly appreciate any feedback.

Thanks everyone,
Kyle

[John Oreopoulos]

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Kyle, that's a great question, and I too would like to know the full answer to this. In general they are not coplanar, but I would have said the opposite to what you stated below for TIRF objectives. For NA greater than 1.4, the back focal plane is usually somewhere inside the objective and NOT near the back edge of the objective barrel. I know this for two reasons:

1. A Zemax mode of a TIRF objective I have shows that you get a collimated beam coming out of the objective only when the light coming through the back is focused to a plane approximately midway the axial length of the objective.

2. In real life, you can setup the same situation. Then remove the objective from the microscope nosepiece and hold a tissue paper in the empty space where the TIRF objective was. You'll find as you run the tissue paper up and down the space, that somewhere above the nosepiece position the laser light will come to a focus which would have been inside the objective.

The specific objective that I have used in my model and in real life is a 60x Nikon TIRF 1.49 NA objective lens, so it may be manufacture dependent.

Sincerely,

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2015-12-22, at 12:19 PM, Douglass Kyle Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
> I'm making an illustration of a microscope objective to demonstrate important concepts in through-the-objective TIRF illumination and ran into a question that I haven't been able to find an answer for.
>
> For a standard, infinity-corrected objective, are there any rules for determining the location of the exit pupil relative to the back focal plane? Is it manufacturer-dependent? I know that the two planes are not coplanar for any general optical system, but in my experience with 100X, NA >= 1.4 oil objectives the two planes are pretty close to one another, lying somewhere near the back edge of the objective barrel.
>
> Would it be "incorrect" to illustrate them as coplanar? I'd greatly appreciate any feedback.
>
> Thanks everyone,
> Kyle

[Zdenek Svindrych-2]

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Yes,
I've made the same observation. With low-power (Olympus) objective the BFP
(the plane at which the incoming beam should be focused to achieve
collimated output beam) is near the objective flange, but with higher power
(40x, 60x) it's midway the objective height or even a bit closer to the
sample (60x / 1.45 NA, TIRFM). Olympus even distinguishes the BFP position
of low power objectives (described as "NORMAL") and high power objectives
(labelled "BFP 1"), and some (Olympus) illuminators and DIC holders can be
switched between those two positions.
Note also that microscope objectives are not designed to produce collimated
beam on the sample side, though it works fine in TIRF, where you use only
tiny part of the objective NA. Also, the main objective design parameters
are magnification, parfocal distance and various optical corrections, so I
assume the BFP position would differ between objective models and
manufacturers...

Best, zdenek

--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, VA
http://www.kcci.virginia.edu/
tel: [phone]


---------- Původní zpráva ----------
Od: John Oreopoulos <[hidden email]>
Komu: [hidden email]
Datum: 22. 12. 2015 12:43:53
Předmět: Re: Objective back focal plane vs. exit pupil plane locations

"*****
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Kyle, that's a great question, and I too would like to know the full answer
to this. In general they are not coplanar, but I would have said the
opposite to what you stated below for TIRF objectives. For NA greater than
1.4, the back focal plane is usually somewhere inside the objective and NOT
near the back edge of the objective barrel. I know this for two reasons:

1. A Zemax mode of a TIRF objective I have shows that you get a collimated
beam coming out of the objective only when the light coming through the back
is focused to a plane approximately midway the axial length of the
objective.

2. In real life, you can setup the same situation. Then remove the objective
from the microscope nosepiece and hold a tissue paper in the empty space
where the TIRF objective was. You'll find as you run the tissue paper up and
down the space, that somewhere above the nosepiece position the laser light
will come to a focus which would have been inside the objective.

The specific objective that I have used in my model and in real life is a 60
x Nikon TIRF 1.49 NA objective lens, so it may be manufacture dependent.

Sincerely,

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca



On 2015-12-22, at 12:19 PM, Douglass Kyle Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hi all,
> I'm making an illustration of a microscope objective to demonstrate
important concepts in through-the-objective TIRF illumination and ran into a
question that I haven't been able to find an answer for.
>
> For a standard, infinity-corrected objective, are there any rules for
determining the location of the exit pupil relative to the back focal plane?
Is it manufacturer-dependent? I know that the two planes are not coplanar
for any general optical system, but in my experience with 100X, NA >= 1.4
oil objectives the two planes are pretty close to one another, lying
somewhere near the back edge of the objective barrel.
>
> Would it be "incorrect" to illustrate them as coplanar? I'd greatly
appreciate any feedback.
>
> Thanks everyone,
> Kyle"

[Michael Giacomelli]

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*****

If you email Zeiss and are persistent, I've had good luck getting them
to give me actual mechanical diagrams with the BFPs marked for their
objectives.  I'd post the diagrams here, but they actually stamped the
"confidential" and they explicitly asked me not to share them.  I have
no idea why they are so protective; anyone with the objective and a
lens can measure it fairly accurately by building a telescope.

For the low NA objectives (<0.15) I asked about, the BFP is a
centimeter or two above the threading.  For the single high NA
immersion objective I inquired about, BFP is exactly the point where
the threading met the objective housing.  Just assuming the BFP and
exit pupil are the base of the threading is probably a good
approximation, but if you're designing a system where its critical
(e.g. scanning confocal), I'd suggest emailing them and asking.

Mike

On Tue, Dec 22, 2015 at 1:29 PM, Zdenek Svindrych <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Yes,
> I've made the same observation. With low-power (Olympus) objective the BFP
> (the plane at which the incoming beam should be focused to achieve
> collimated output beam) is near the objective flange, but with higher power
> (40x, 60x) it's midway the objective height or even a bit closer to the
> sample (60x / 1.45 NA, TIRFM). Olympus even distinguishes the BFP position
> of low power objectives (described as "NORMAL") and high power objectives
> (labelled "BFP 1"), and some (Olympus) illuminators and DIC holders can be
> switched between those two positions.
> Note also that microscope objectives are not designed to produce collimated
> beam on the sample side, though it works fine in TIRF, where you use only
> tiny part of the objective NA. Also, the main objective design parameters
> are magnification, parfocal distance and various optical corrections, so I
> assume the BFP position would differ between objective models and
> manufacturers...
>
> Best, zdenek
>
> --
> Zdenek Svindrych, Ph.D.
> W.M. Keck Center for Cellular Imaging (PLSB 003)
> University of Virginia, Charlottesville, VA
> http://www.kcci.virginia.edu/
> tel: [phone]
>
>
> ---------- Původní zpráva ----------
> Od: John Oreopoulos <[hidden email]>
> Komu: [hidden email]
> Datum: 22. 12. 2015 12:43:53
> Předmět: Re: Objective back focal plane vs. exit pupil plane locations
>
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Kyle, that's a great question, and I too would like to know the full answer
> to this. In general they are not coplanar, but I would have said the
> opposite to what you stated below for TIRF objectives. For NA greater than
> 1.4, the back focal plane is usually somewhere inside the objective and NOT
> near the back edge of the objective barrel. I know this for two reasons:
>
> 1. A Zemax mode of a TIRF objective I have shows that you get a collimated
> beam coming out of the objective only when the light coming through the back
> is focused to a plane approximately midway the axial length of the
> objective.
>
> 2. In real life, you can setup the same situation. Then remove the objective
> from the microscope nosepiece and hold a tissue paper in the empty space
> where the TIRF objective was. You'll find as you run the tissue paper up and
> down the space, that somewhere above the nosepiece position the laser light
> will come to a focus which would have been inside the objective.
>
> The specific objective that I have used in my model and in real life is a 60
> x Nikon TIRF 1.49 NA objective lens, so it may be manufacture dependent.
>
> Sincerely,
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research Inc.
> A Division of Andor Technology
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
>
> On 2015-12-22, at 12:19 PM, Douglass Kyle Michael wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> Hi all,
>> I'm making an illustration of a microscope objective to demonstrate
> important concepts in through-the-objective TIRF illumination and ran into a
> question that I haven't been able to find an answer for.
>>
>> For a standard, infinity-corrected objective, are there any rules for
> determining the location of the exit pupil relative to the back focal plane?
> Is it manufacturer-dependent? I know that the two planes are not coplanar
> for any general optical system, but in my experience with 100X, NA >= 1.4
> oil objectives the two planes are pretty close to one another, lying
> somewhere near the back edge of the objective barrel.
>>
>> Would it be "incorrect" to illustrate them as coplanar? I'd greatly
> appreciate any feedback.
>>
>> Thanks everyone,
>> Kyle"

[Reto Fiolka]

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*****

Hi Kyle

I normally map out the location of the BFP empirically, and it usually lies inside the
objective. I thought maybe some here on the list would be interested how to do that.

Here is what I came up, I am eager to learn other methods:

For confocal microscope- type beam scanning (i.e. a laser focus in the front focal plane),
I usually determine the BFP position by determining where the scanning mirror needs to
be conjugated to obtain a telecentric scan, i.e. the focused beam translates laterally
without changing its angle. Off course, the mirror relay telescope needs to be telecentric
to start with.

To find the BFP, I put a sinusoidal signal on the scan mirror to let it oscillate between the
scan range limits. I then translate the objective slowly along its optical axis and observe
with a paper card the diverging cone of the laser beam, as it emerges from the focal
plane. If your mirror is not conjugated to the BFP, the laser cone will wobble (as it is not
doing a pure translation, but also some angular steering). Once you hit the BFP, the laser
cone looks like not moving at all (as it only move some 100 microns laterally, depending
on scan range, too small to see by eye).

The inverse process, i.e. in a TIRF objective varying the incidence angle by laterally
scanning the spot in the BFP yielded often a less clear result. Ideally, the beam should be
collimated and remain centered in the sample plane and not move when varying
incidence angle. This was to me often hard to achieve.

But as Zdenek wrote, going the "wrong way" through the objective at its periphery is a bit
dicey

Best,
Reto

[Kyle Michael Douglass]

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Thanks everyone for their input to this question.

Here's a quick followup: is the exit pupil usually the rear-most aperture, i.e. coplanar with the threads? I am not in the lab right now to check, but I recall looking through the rear of the barrel and noticing that the image filled the entire aperture, which to me suggests that the rear, hard aperture is indeed the exit pupil. Also, I measured the rear aperture size on two different non-TIRF Nikon CFI60 objectives and it matches the theoretical results: D = 2*NA*f.

The reason I ask is because it would seem like an offset between the exit pupil and back focal plane could result in a partial truncation of the beam when bringing it to a focus in the back focal plane. If this were true, then the ideal case for TIRF would be to make them coplanar to avoid blocking part of the beam at the edge of the exit pupil.

Thanks again!
Kyle
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Reto Fiolka [[hidden email]]
Sent: Tuesday, December 22, 2015 7:58 PM
To: [hidden email]
Subject: Re: Objective back focal plane vs. exit pupil plane locations

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

Hi Kyle

I normally map out the location of the BFP empirically, and it usually lies inside the
objective. I thought maybe some here on the list would be interested how to do that.

Here is what I came up, I am eager to learn other methods:

For confocal microscope- type beam scanning (i.e. a laser focus in the front focal plane),
I usually determine the BFP position by determining where the scanning mirror needs to
be conjugated to obtain a telecentric scan, i.e. the focused beam translates laterally
without changing its angle. Off course, the mirror relay telescope needs to be telecentric
to start with.

To find the BFP, I put a sinusoidal signal on the scan mirror to let it oscillate between the
scan range limits. I then translate the objective slowly along its optical axis and observe
with a paper card the diverging cone of the laser beam, as it emerges from the focal
plane. If your mirror is not conjugated to the BFP, the laser cone will wobble (as it is not
doing a pure translation, but also some angular steering). Once you hit the BFP, the laser
cone looks like not moving at all (as it only move some 100 microns laterally, depending
on scan range, too small to see by eye).

The inverse process, i.e. in a TIRF objective varying the incidence angle by laterally
scanning the spot in the BFP yielded often a less clear result. Ideally, the beam should be
collimated and remain centered in the sample plane and not move when varying
incidence angle. This was to me often hard to achieve.

But as Zdenek wrote, going the "wrong way" through the objective at its periphery is a bit
dicey

Best,
Reto