field flatness problem (wide field)

classic Classic list List threaded Threaded
5 messages Options
David Lenzi David Lenzi
Reply | Threaded
Open this post in threaded view
|

field flatness problem (wide field)

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

Hi all,
I am trying to solve a field flatness issue, and was wondering if anyone else
had come across something similar.  If focussed at the center of the field, the
edges are out of focus, and vice versa, with the difference in z about 0.5 to
1.0 um,  the center lower than the edges.  The microscope is a Leica DMI6000B
(inverted), and the objective is a 63x/1.4NA plan apo oil immersion.  The
camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), which
captures a field of view of 21.7 mm (on the diagonal) at the objective's back
aperture.  I'm still waiting to get an answer from Leica on what the flatness
specification is, but it seems to me it should be better than this.

Here is what else I know:
- i don't think it is the stage because the effect is radially symmetrical, and
object go in and out of focus as they are moved from one corner, through the
center, and to the opposite corner of the field.
- The effect is independent of wavelength or filter cube.
- The effect is also visible at 40x/1.3 NA (oil) and 20x (air).

- The other key test is whether the effect is visible at the camera as well as
through the eye pieces. This is harder to tell, but I think it is visible through
the eye pieces.

Any suggestions appreciated!   -David Lenzi
JOEL B. SHEFFIELD JOEL B. SHEFFIELD
Reply | Threaded
Open this post in threaded view
|

Re: field flatness problem (wide field)

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

HI David,

I can't tell from your note whether your camera has a relay lens of some
sort.  Frequently, these lenses are a source of this kind of curvature of
the field.  Have you checked the camera itself?

Joel


On Thu, May 31, 2012 at 3:11 PM, David Lenzi <[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 am trying to solve a field flatness issue, and was wondering if anyone
> else
> had come across something similar.  If focussed at the center of the
> field, the
> edges are out of focus, and vice versa, with the difference in z about 0.5
> to
> 1.0 um,  the center lower than the edges.  The microscope is a Leica
> DMI6000B
> (inverted), and the objective is a 63x/1.4NA plan apo oil immersion.  The
> camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels),
> which
> captures a field of view of 21.7 mm (on the diagonal) at the objective's
> back
> aperture.  I'm still waiting to get an answer from Leica on what the
> flatness
> specification is, but it seems to me it should be better than this.
>
> Here is what else I know:
> - i don't think it is the stage because the effect is radially
> symmetrical, and
> object go in and out of focus as they are moved from one corner, through
> the
> center, and to the opposite corner of the field.
> - The effect is independent of wavelength or filter cube.
> - The effect is also visible at 40x/1.3 NA (oil) and 20x (air).
>
> - The other key test is whether the effect is visible at the camera as
> well as
> through the eye pieces. This is harder to tell, but I think it is visible
> through
> the eye pieces.
>
> Any suggestions appreciated!   -David Lenzi
>



--


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [hidden email]
URL:  http://astro.temple.edu/~jbs
Gregg Jarvis Gregg Jarvis
Reply | Threaded
Open this post in threaded view
|

Re: field flatness problem (wide field)

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

David,
Do you have any other microscopes available to see if it is in fact the
microscope?
It seems it could be the slide depth/poor slide? or the microscope
objective is out of alignment or not seated all the way into the microscope
housing,"spherical aberration"
did someone bump it or change out the objectives recently?

On Thu, May 31, 2012 at 3:11 PM, David Lenzi <[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 am trying to solve a field flatness issue, and was wondering if anyone
> else
> had come across something similar.  If focussed at the center of the
> field, the
> edges are out of focus, and vice versa, with the difference in z about 0.5
> to
> 1.0 um,  the center lower than the edges.  The microscope is a Leica
> DMI6000B
> (inverted), and the objective is a 63x/1.4NA plan apo oil immersion.  The
> camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels),
> which
> captures a field of view of 21.7 mm (on the diagonal) at the objective's
> back
> aperture.  I'm still waiting to get an answer from Leica on what the
> flatness
> specification is, but it seems to me it should be better than this.
>
> Here is what else I know:
> - i don't think it is the stage because the effect is radially
> symmetrical, and
> object go in and out of focus as they are moved from one corner, through
> the
> center, and to the opposite corner of the field.
> - The effect is independent of wavelength or filter cube.
> - The effect is also visible at 40x/1.3 NA (oil) and 20x (air).
>
> - The other key test is whether the effect is visible at the camera as
> well as
> through the eye pieces. This is harder to tell, but I think it is visible
> through
> the eye pieces.
>
> Any suggestions appreciated!   -David Lenzi
>



--
Gregg Jarvis
Advanced Products Group
Omega Optical Inc.
24 Omega Drive
Brattleboro, VT 05301
[hidden email]
1-802-251-7316
John Oreopoulos John Oreopoulos
Reply | Threaded
Open this post in threaded view
|

Re: field flatness problem (wide field)

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

Hi David,

My guess is the following: Since you're now using a CMOS camera with a much larger chip area, you're probably seeing the field curvature that was always there associated with the objective and tube lens of your microscope system. You probably didn't see it before simply because the camera you were using previously could only view a much smaller portion of the entire field of view presented by the objective and tube lens combination. One way to test that theory would be to switch back to your older camera and physically translate it across the field of view and see if you observe the same effect as before with the other camera. Instead of using a camera hard mount, put the camera on a lab jack and place a dark cloak over the camera the microscope side port when translating.

John Oreopoulos


On 2012-05-31, at 3:11 PM, David Lenzi wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
> I am trying to solve a field flatness issue, and was wondering if anyone else
> had come across something similar.  If focussed at the center of the field, the
> edges are out of focus, and vice versa, with the difference in z about 0.5 to
> 1.0 um,  the center lower than the edges.  The microscope is a Leica DMI6000B
> (inverted), and the objective is a 63x/1.4NA plan apo oil immersion.  The
> camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), which
> captures a field of view of 21.7 mm (on the diagonal) at the objective's back
> aperture.  I'm still waiting to get an answer from Leica on what the flatness
> specification is, but it seems to me it should be better than this.
>
> Here is what else I know:
> - i don't think it is the stage because the effect is radially symmetrical, and
> object go in and out of focus as they are moved from one corner, through the
> center, and to the opposite corner of the field.
> - The effect is independent of wavelength or filter cube.
> - The effect is also visible at 40x/1.3 NA (oil) and 20x (air).
>
> - The other key test is whether the effect is visible at the camera as well as
> through the eye pieces. This is harder to tell, but I think it is visible through
> the eye pieces.
>
> Any suggestions appreciated!   -David Lenzi
George McNamara George McNamara
Reply | Threaded
Open this post in threaded view
|

Re: field flatness problem (wide field)

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

Hi David,

I was working with a Leica plan apo 63x/1.4 NA oil immersion objective
lens on our SP5 confocal microscope (DMI6000). The user's specimens were
flat cells on coverglasses - each coverglass had little specks of green
thingies strewn over it (probably residue of cells that had mostly been
blown off during sample prep).

All the coverglasses had tiny specks that were in focus at the
coverglass over the entire field of view of the eyepiece. Yes, my eyes
are not calibrated in Z. Some suggestions:

is there some optical component in the light path that is not needed - a
0.5x or 1.6x relay lens (in camera mount) or optovar (in the stand) for
example?
   ... if yes, is it designed for this microscope?
   ... sometimes microscope components stop working - is it possible a
motorized turret that is supposed to remove a relay lens is stuck in the
wrong place?
when you look at the same specimens on a different microscope with
comparable lens - plan apo 63x or 60x 1.4 NA - is it flat or
pincushioned? (especailly same lens on another DMI6000).
are you looking at features directly at the coverglass ... is the
coverglass 170 um thick (or at least out of a box that says #1.5)? ...
do multiple specimens behave the same way?
   ... if you are looking at something far away, say cells on a Lab-Tek
chamber slide with 50 um gap to the coverglass, this lens is designed to
image at the [170 um thick] coverglass, not far (10+ um) away.
are you using the right immersion oil? (though you mentioned your 20x
dry lens has the same problems)
is your lens "messed up"? (i.e. if you look at the front element on a
stereomicroscope, does it look ok or "messed up")


George


On 5/31/2012 3:11 PM, David Lenzi wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
> I am trying to solve a field flatness issue, and was wondering if anyone else
> had come across something similar.  If focussed at the center of the field, the
> edges are out of focus, and vice versa, with the difference in z about 0.5 to
> 1.0 um,  the center lower than the edges.  The microscope is a Leica DMI6000B
> (inverted), and the objective is a 63x/1.4NA plan apo oil immersion.  The
> camera we're using has a large chip (Fairchild CMOS, 2560 x 2160 pixels), which
> captures a field of view of 21.7 mm (on the diagonal) at the objective's back
> aperture.  I'm still waiting to get an answer from Leica on what the flatness
> specification is, but it seems to me it should be better than this.
>
> Here is what else I know:
> - i don't think it is the stage because the effect is radially symmetrical, and
> object go in and out of focus as they are moved from one corner, through the
> center, and to the opposite corner of the field.
> - The effect is independent of wavelength or filter cube.
> - The effect is also visible at 40x/1.3 NA (oil) and 20x (air).
>
> - The other key test is whether the effect is visible at the camera as well as
> through the eye pieces. This is harder to tell, but I think it is visible through
> the eye pieces.
>
> Any suggestions appreciated!   -David Lenzi
>
>