Bad fluorescence uniformity across the field of view with a Yokogawa W1 unit

[Emmanuel Levy]

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Dear All,

We just acquired a Yokogawa W1 system coupled to a X83 Olympus microscope.

It is working fine. However, we noticed significant non-uniformity across
the field of view. We are using sCMOS cameras (flash4) that have a large
chip, but the W1 was designed specifically for such a large field of view
so I doubt that the non uniformity we observe is normal. Hence I'd like to
ask if anyone encountered a similar problem, and most importantly, of the
same magnitude.

To give specific numbers, you can see below a table reflecting the
intensity that we measured for a specific object (cell), which we moved
from the center to all corners of the image. The first thing one can notice
is that the laser is not centered but considering the differences observed
(over 80%!) this would not improve things much even if it was.

Fluorescence intensity of an object moved in different parts of the field
of view:
---------------------------------
|         Left    Center   Right|
---------------------------------
| Top     386     1110     760  |
| Center  540     1120     1086 |
| Bottom  200     450      275  |
---------------------------------

We see the same effect with both 488 and 561 lasers.

If you have any experience with such an issue I'd be grateful to hear about
it, and hopefully how it may be fixed.

Thank you,

Emmanuel

[Zdenek Svindrych-2]

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Hi Emmanuel,
what objective are you using? Maybe the objective has smaller FOV, or the
aberrations at the edge of FOV are so severe that the fluorescence can't
make it back through the pinholes...

What sample are you using? Is it thin fluorescent layer or 'sea of
fluorescence'?

I have only worked with CSU X1 (with 8mm CCD), the uniformity was far from
perfect, but much better than yours. Also some systems seem to be tuned
better than others. With 'UPlanSApo' objectives you should be able to get
better results. Ask Andor!

They may offer you a (not quite cheap) upgrade called Borealis, a simple
setup with multimode fibers (instead if singlemode), vibrating homogenizer
and critical illumination setup (instead of Kohler-like). I liked this
illumination style very much in Vutara superresolution scopes, though I
haven't tried it with Andor's confocals.

---- absolutely no commercial interest ------

Best, zdenek, kcci.virginia.edu






---------- Původní zpráva ----------
Od: Emmanuel Levy <[hidden email]>
Komu: [hidden email]
Datum: 13. 12. 2014 12:29:45
Předmět: Bad fluorescence uniformity across the field of view with a
Yokogawa W1 unit

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

Dear All,

We just acquired a Yokogawa W1 system coupled to a X83 Olympus microscope.

It is working fine. However, we noticed significant non-uniformity across
the field of view. We are using sCMOS cameras (flash4) that have a large
chip, but the W1 was designed specifically for such a large field of view
so I doubt that the non uniformity we observe is normal. Hence I'd like to
ask if anyone encountered a similar problem, and most importantly, of the
same magnitude.

To give specific numbers, you can see below a table reflecting the
intensity that we measured for a specific object (cell), which we moved
from the center to all corners of the image. The first thing one can notice
is that the laser is not centered but considering the differences observed
(over 80%!) this would not improve things much even if it was.

Fluorescence intensity of an object moved in different parts of the field
of view:
---------------------------------
| Left Center Right|
---------------------------------
| Top 386 1110 760 |
| Center 540 1120 1086 |
| Bottom 200 450 275 |
---------------------------------

We see the same effect with both 488 and 561 lasers.

If you have any experience with such an issue I'd be grateful to hear about
it, and hopefully how it may be fixed.

Thank you,

Emmanuel"

[Emmanuel Levy]

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

For these measurements we used a 60X UplanApo, 1.35NA so I do not believe
that the objective is the problem. The Borealis option does not exist for
the W1 but it does suggest that things could be improved.

If there are other users of the W1, I would be keen to know what values you
get.

The sample observed is a specific yeast cell tagged with YFP, we avoided
the Chroma fluorescent slides as we read that it was not optimal to check
uniformity. I should mention that under the conditions tested bleaching was
negligible (the "center" positioning was measured at the beginning and at
the end of all measurements, and gave the same value).

A colleague suggested that the laser was not properly focused at the back
focal plane on the objective. But I cannot check this as I cannot open the
W1 box myself (or I'd loose the warranty).

Thanks for your feedback,

Emmanuel


On 13 December 2014 at 20:36, 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.
> *****
>
> Hi Emmanuel,
> what objective are you using? Maybe the objective has smaller FOV, or the
> aberrations at the edge of FOV are so severe that the fluorescence can't
> make it back through the pinholes...
>
> What sample are you using? Is it thin fluorescent layer or 'sea of
> fluorescence'?
>
> I have only worked with CSU X1 (with 8mm CCD), the uniformity was far from
> perfect, but much better than yours. Also some systems seem to be tuned
> better than others. With 'UPlanSApo' objectives you should be able to get
> better results. Ask Andor!
>
> They may offer you a (not quite cheap) upgrade called Borealis, a simple
> setup with multimode fibers (instead if singlemode), vibrating homogenizer
> and critical illumination setup (instead of Kohler-like). I liked this
> illumination style very much in Vutara superresolution scopes, though I
> haven't tried it with Andor's confocals.
>
> ---- absolutely no commercial interest ------
>
> Best, zdenek, kcci.virginia.edu
>
>
>
>
>
>
> ---------- Původní zpráva ----------
> Od: Emmanuel Levy <[hidden email]>
> Komu: [hidden email]
> Datum: 13. 12. 2014 12:29:45
> Předmět: Bad fluorescence uniformity across the field of view with a
> Yokogawa W1 unit
>
> "*****
> 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.
> *****
>
> Dear All,
>
> We just acquired a Yokogawa W1 system coupled to a X83 Olympus microscope.
>
> It is working fine. However, we noticed significant non-uniformity across
> the field of view. We are using sCMOS cameras (flash4) that have a large
> chip, but the W1 was designed specifically for such a large field of view
> so I doubt that the non uniformity we observe is normal. Hence I'd like to
> ask if anyone encountered a similar problem, and most importantly, of the
> same magnitude.
>
> To give specific numbers, you can see below a table reflecting the
> intensity that we measured for a specific object (cell), which we moved
> from the center to all corners of the image. The first thing one can notice
> is that the laser is not centered but considering the differences observed
> (over 80%!) this would not improve things much even if it was.
>
> Fluorescence intensity of an object moved in different parts of the field
> of view:
> ---------------------------------
> | Left Center Right|
> ---------------------------------
> | Top 386 1110 760 |
> | Center 540 1120 1086 |
> | Bottom 200 450 275 |
> ---------------------------------
>
> We see the same effect with both 488 and 561 lasers.
>
> If you have any experience with such an issue I'd be grateful to hear about
> it, and hopefully how it may be fixed.
>
> Thank you,
>
> Emmanuel"
>

[Michael Giacomelli]

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

Hi Emmanuel,

I'm not familiar with that specific scan unit, but I have aligned
scanning system before, and excessive vignetting at large scan angles often
means that the scanners are axially displaced from the back aperture of the
scan lens (or tube lens if the scan lens is integrated into the scanner).
This results in reduced back-coupling into the pinhole off axis, but
relatively normal coupling on-axis.

The other possibility is just that your objective or scanner do not perform
well, but if you are seeing significant loss of signal at reasonable field
(say half the field number or less), I would try to check the alignment.

Mike

On Sat, Dec 13, 2014 at 2:22 PM, Emmanuel Levy <[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.
> *****
>
> Dear Zdenek,
>
> For these measurements we used a 60X UplanApo, 1.35NA so I do not believe
> that the objective is the problem. The Borealis option does not exist for
> the W1 but it does suggest that things could be improved.
>
> If there are other users of the W1, I would be keen to know what values you
> get.
>
> The sample observed is a specific yeast cell tagged with YFP, we avoided
> the Chroma fluorescent slides as we read that it was not optimal to check
> uniformity. I should mention that under the conditions tested bleaching was
> negligible (the "center" positioning was measured at the beginning and at
> the end of all measurements, and gave the same value).
>
> A colleague suggested that the laser was not properly focused at the back
> focal plane on the objective. But I cannot check this as I cannot open the
> W1 box myself (or I'd loose the warranty).
>
> Thanks for your feedback,
>
> Emmanuel
>
>
> On 13 December 2014 at 20:36, 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.
> > *****
> >
> > Hi Emmanuel,
> > what objective are you using? Maybe the objective has smaller FOV, or the
> > aberrations at the edge of FOV are so severe that the fluorescence can't
> > make it back through the pinholes...
> >
> > What sample are you using? Is it thin fluorescent layer or 'sea of
> > fluorescence'?
> >
> > I have only worked with CSU X1 (with 8mm CCD), the uniformity was far
> from
> > perfect, but much better than yours. Also some systems seem to be tuned
> > better than others. With 'UPlanSApo' objectives you should be able to get
> > better results. Ask Andor!
> >
> > They may offer you a (not quite cheap) upgrade called Borealis, a simple
> > setup with multimode fibers (instead if singlemode), vibrating
> homogenizer
> > and critical illumination setup (instead of Kohler-like). I liked this
> > illumination style very much in Vutara superresolution scopes, though I
> > haven't tried it with Andor's confocals.
> >
> > ---- absolutely no commercial interest ------
> >
> > Best, zdenek, kcci.virginia.edu
> >
> >
> >
> >
> >
> >
> > ---------- Původní zpráva ----------
> > Od: Emmanuel Levy <[hidden email]>
> > Komu: [hidden email]
> > Datum: 13. 12. 2014 12:29:45
> > Předmět: Bad fluorescence uniformity across the field of view with a
> > Yokogawa W1 unit
> >
> > "*****
> > 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.
> > *****
> >
> > Dear All,
> >
> > We just acquired a Yokogawa W1 system coupled to a X83 Olympus
> microscope.
> >
> > It is working fine. However, we noticed significant non-uniformity across
> > the field of view. We are using sCMOS cameras (flash4) that have a large
> > chip, but the W1 was designed specifically for such a large field of view
> > so I doubt that the non uniformity we observe is normal. Hence I'd like
> to
> > ask if anyone encountered a similar problem, and most importantly, of the
> > same magnitude.
> >
> > To give specific numbers, you can see below a table reflecting the
> > intensity that we measured for a specific object (cell), which we moved
> > from the center to all corners of the image. The first thing one can
> notice
> > is that the laser is not centered but considering the differences
> observed
> > (over 80%!) this would not improve things much even if it was.
> >
> > Fluorescence intensity of an object moved in different parts of the field
> > of view:
> > ---------------------------------
> > | Left Center Right|
> > ---------------------------------
> > | Top 386 1110 760 |
> > | Center 540 1120 1086 |
> > | Bottom 200 450 275 |
> > ---------------------------------
> >
> > We see the same effect with both 488 and 561 lasers.
> >
> > If you have any experience with such an issue I'd be grateful to hear
> about
> > it, and hopefully how it may be fixed.
> >
> > Thank you,
> >
> > Emmanuel"
> >
>

[James Pawley]

*****
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.
*****

Dear Emmanuel,

The fact that your intensity pattern is
asymmetric says to me that the system may have an
alignment problem (emitting end of the laser
fibre not exactly on axis or the coupling optics
not lined up with the optical axis of the
objective etc. ...) . I suggest that you turn on
the system with no specimen,  and hold a white
card say 6-10 cm from the objective. If the BFP
is properly illuminated, you should see a FULL,
UNIFORM circle of excitation light and the circle
should get larger (and dimmer) as you move the
card away from the objective.

Another problem might be the coupling optics
between the disk and the CMOS. It needs to pass
any light that makes it through the pinholes on
to the chip. Vignetting here will make the
corners darker. Talk to Yokogawa to make sure you
have the proper lens.

What is the specimen? If it is planar (and thin)
you will need a "Plan"  objective to keep it in
focus. You mention moving the same object to 9
positions in the FOV. Are you sure it didn't
bleach during this process? if your specimen is
thick and your lens is not a "Plan" lens, then
the CMOS will be focused on a curved surface in
the specimen. If the RI of everything (embedment,
coverslip, immersion oil or other immersion
liquid) isn't exactly correct, you will have
different amounts of spherical aberration
throughout the field of view and this is a great
way to lose signal.

Finally, as shown in Chapter 11, the performance
of even very expensive optics (Zeiss 1.2 40x
cPlanAPO, or 1.4 plan APO oil) drops off as one
moves to the edge of the FOV. At the very least
this is caused by vignetting. (Some of the
high-NA rays emerging from the near the edge of
the FOV just "hit the wall". (The glass just
isn't large enough in diameter. Often this is
limited to the available diameter of the threaded
hole in the objective nosepiece.)

Cheers,

Jim P.

>*****
>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 Emmanuel, what objective are you using? Maybe
>the objective has smaller FOV, or the
>aberrations at the edge of FOV are so severe
>that the fluorescence can't make it back through
>the pinholes... What sample are you using? Is it
>thin fluorescent layer or 'sea of fluorescence'?
>I have only worked with CSU X1 (with 8mm CCD),
>the uniformity was far from perfect, but much
>better than yours. Also some systems seem to be
>tuned better than others. With 'UPlanSApo'
>objectives you should be able to get better
>results. Ask Andor! They may offer you a (not
>quite cheap) upgrade called Borealis, a simple
>setup with multimode fibers (instead if
>singlemode), vibrating homogenizer and critical
>illumination setup (instead of Kohler-like). I
>liked this illumination style very much in
>Vutara superresolution scopes, though I haven't
>tried it with Andor's confocals. ---- absolutely
>no commercial interest ------ Best, zdenek,
>kcci.virginia.edu ---------- PÛvodní zpráva
>---------- Od: Emmanuel Levy
><[hidden email]> Komu:
>[hidden email] Datum: 13. 12.
>2014 12:29:45 PÞedmût: Bad fluorescence
>uniformity across the field of view with a
>Yokogawa W1 unit "***** 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. ***** Dear All, We
>just acquired a Yokogawa W1 system coupled to a
>X83 Olympus microscope. It is working fine.
>However, we noticed significant non-uniformity
>across the field of view. We are using sCMOS
>cameras (flash4) that have a large chip, but the
>W1 was designed specifically for such a large
>field of view so I doubt that the non uniformity
>we observe is normal. Hence I'd like to ask if
>anyone encountered a similar problem, and most
>importantly, of the same magnitude. To give
>specific numbers, you can see below a table
>reflecting the intensity that we measured for a
>specific object (cell), which we moved from the
>center to all corners of the image. The first
>thing one can notice is that the laser is not
>centered but considering the differences
>observed (over 80%!) this would not improve
>things much even if it was. Fluorescence
>intensity of an object moved in different parts
>of the field of view:
>--------------------------------- | Left Center
>Right| --------------------------------- | Top
>386 1110 760 | | Center 540 1120 1086 | | Bottom
>200 450 275 | ---------------------------------
>We see the same effect with both 488 and 561
>lasers. If you have any experience with such an
>issue I'd be grateful to hear about it, and
>hopefully how it may be fixed. Thank you,
>Emmanuel"

--
               ****************************************
James and Christine Pawley, 5446 Burley Place (PO
Box 2348), Sechelt, BC, Canada, V0N3A0,
Phone [phone], email <[hidden email]>
NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!) 1-604-989-6146

[Emmanuel Levy]

*****
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.
*****

Dear All,

Thank you for your feedback, I would like to address a few points that were
raised:

- The objective is 60X oil, plan Apo
- The sample is thin and did not bleach
- The laser is indeed not perfectly aligned but I do not think it could
explain the >80% variation seen across the FOV.

One important addition: this problem is absent in brightfield (even with
the disk engaged of course). Therefore, the field number should be OK along
the light path. To me, this confirms that the problem is in the
illumination and not the collection.

If there are W1 users, I'd be really grateful if you could share your
experience.

Thank you,

Emmanuel



On 14 December 2014 at 03:46, James Pawley <[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.
> *****
>
> Dear Emmanuel,
>
> The fact that your intensity pattern is asymmetric says to me that the
> system may have an alignment problem (emitting end of the laser fibre not
> exactly on axis or the coupling optics not lined up with the optical axis
> of the objective etc. ...) . I suggest that you turn on the system with no
> specimen,  and hold a white card say 6-10 cm from the objective. If the BFP
> is properly illuminated, you should see a FULL, UNIFORM circle of
> excitation light and the circle should get larger (and dimmer) as you move
> the card away from the objective.
>
> Another problem might be the coupling optics between the disk and the
> CMOS. It needs to pass any light that makes it through the pinholes on to
> the chip. Vignetting here will make the corners darker. Talk to Yokogawa to
> make sure you have the proper lens.
>
> What is the specimen? If it is planar (and thin) you will need a "Plan"
> objective to keep it in focus. You mention moving the same object to 9
> positions in the FOV. Are you sure it didn't bleach during this process? if
> your specimen is thick and your lens is not a "Plan" lens, then the CMOS
> will be focused on a curved surface in the specimen. If the RI of
> everything (embedment, coverslip, immersion oil or other immersion liquid)
> isn't exactly correct, you will have different amounts of spherical
> aberration throughout the field of view and this is a great way to lose
> signal.
>
> Finally, as shown in Chapter 11, the performance of even very expensive
> optics (Zeiss 1.2 40x cPlanAPO, or 1.4 plan APO oil) drops off as one moves
> to the edge of the FOV. At the very least this is caused by vignetting.
> (Some of the high-NA rays emerging from the near the edge of the FOV just
> "hit the wall". (The glass just isn't large enough in diameter. Often this
> is limited to the available diameter of the threaded hole in the objective
> nosepiece.)
>
> Cheers,
>
> Jim P.
>
>
>  *****
>> 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 Emmanuel, what objective are you using? Maybe the objective has
>> smaller FOV, or the aberrations at the edge of FOV are so severe that the
>> fluorescence can't make it back through the pinholes... What sample are you
>> using? Is it thin fluorescent layer or 'sea of fluorescence'? I have only
>> worked with CSU X1 (with 8mm CCD), the uniformity was far from perfect, but
>> much better than yours. Also some systems seem to be tuned better than
>> others. With 'UPlanSApo' objectives you should be able to get better
>> results. Ask Andor! They may offer you a (not quite cheap) upgrade called
>> Borealis, a simple setup with multimode fibers (instead if singlemode),
>> vibrating homogenizer and critical illumination setup (instead of
>> Kohler-like). I liked this illumination style very much in Vutara
>> superresolution scopes, though I haven't tried it with Andor's confocals.
>> ---- absolutely no commercial interest ------ Best, zdenek,
>> kcci.virginia.edu ---------- PÛvodní zpráva ---------- Od: Emmanuel Levy
>> <[hidden email]> Komu: [hidden email] Datum:
>> 13. 12. 2014 12:29:45 PÞedmût: Bad fluorescence uniformity across the field
>> of view with a Yokogawa W1 unit "***** 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. ***** Dear All, We just acquired a Yokogawa W1
>> system coupled to a X83 Olympus microscope. It is working fine. However, we
>> noticed significant non-uniformity across the field of view. We are using
>> sCMOS cameras (flash4) that have a large chip, but the W1 was designed
>> specifically for such a large field of view so I doubt that the non
>> uniformity we observe is normal. Hence I'd like to ask if anyone
>> encountered a similar problem, and most importantly, of the same magnitude.
>> To give specific numbers, you can see below a table reflecting the
>> intensity that we measured for a specific object (cell), which we moved
>> from the center to all corners of the image. The first thing one can notice
>> is that the laser is not centered but considering the differences observed
>> (over 80%!) this would not improve things much even if it was. Fluorescence
>> intensity of an object moved in different parts of the field of view:
>> --------------------------------- | Left Center Right|
>> --------------------------------- | Top 386 1110 760 | | Center 540 1120
>> 1086 | | Bottom 200 450 275 | --------------------------------- We see
>> the same effect with both 488 and 561 lasers. If you have any experience
>> with such an issue I'd be grateful to hear about it, and hopefully how it
>> may be fixed. Thank you, Emmanuel"
>>
>
>
> --
>               ****************************************
> James and Christine Pawley, 5446 Burley Place (PO Box 2348), Sechelt, BC,
> Canada, V0N3A0,
> Phone [phone], email <[hidden email]>
> NEW! NEW! AND DIFFERENT Cell (when I remember to turn it on!)
> 1-604-989-6146
>