Objective phosphorescence

[Benjamin Smith]

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Hey microscopists,
   We observed an odd phenomenon today on a microscope and was wondering if
anyone else has ever seen it.  We were using a DMD do a full field flash
with 420nm light during the flyback of the scanning mirror on a 2P imaging
rig.  We noticed that after the light was turned off, there was a
millisecond long slewing of the signal that looked a lot like
phosphorescence.  In the following image, you can see that the LED is on
for the first portion of the scan, then turns off and the apparent
afterglow: https://goo.gl/2ENHwL

This afterglow was also apparent with an oscilloscope looking at the PMT
and fast mirror signals: https://goo.gl/2AMsvB

   We then systematically removed components from the optical path, and
cleaned everything, and we were eventually able to determine that the glass
in the objective itself was glowing, where if the objective was removed and
the DMD image was shined onto a piece of lens paper or metal, the afterglow
went away:
https://goo.gl/arXYF5
https://goo.gl/cVo2Ev

    The final nail in the coffin to our suspicions was when we then mounted
a plano-convex N-BK7 lens onto the microscope and the effect came back, and
the thicker the lens, the stronger the effect. Also, the effect went away
when we used 540nm light.

With a bit of internet searching I also came across this paper that
confirms there is some visible fluorescence in glass due to trace elements:
http://www.schott.com/d/advanced_optics/87330898-4e56-4d70-965a-3f03c7bc0c80/1.1/schott_tie-36_fluorescence_of_optical_glass_us.pdf

Even when I saw the slew, and the first thing that came to mind was
phosphorescence, the last thing that came to mind was that the glass in the
objective itself was the offender, so I wanted to post this to both give
other people a heads-up, and also to see if anyone else has run into this
phenomenon.

Cheers,
   Ben Smith

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Craig Brideau]

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That is quite an unusual finding, Ben. It would be interesting to try a
fused silica lens to see if that gives the same result or not. Glass can
exhibit all sorts of emissions at shorter wavelength but I have never seen
this particular situation. Some LEDs use fluorescence or phosphorescence in
their emission but you seem to have ruled that out. Fused silica *should*
be pure enough to avoid issues at that wavelength.

Craig


On Nov 20, 2017 8:34 PM, "Benjamin E Smith" <[hidden email]>
wrote:

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*****

Hey microscopists,
   We observed an odd phenomenon today on a microscope and was wondering if
anyone else has ever seen it.  We were using a DMD do a full field flash
with 420nm light during the flyback of the scanning mirror on a 2P imaging
rig.  We noticed that after the light was turned off, there was a
millisecond long slewing of the signal that looked a lot like
phosphorescence.  In the following image, you can see that the LED is on
for the first portion of the scan, then turns off and the apparent
afterglow: https://goo.gl/2ENHwL

This afterglow was also apparent with an oscilloscope looking at the PMT
and fast mirror signals: https://goo.gl/2AMsvB

   We then systematically removed components from the optical path, and
cleaned everything, and we were eventually able to determine that the glass
in the objective itself was glowing, where if the objective was removed and
the DMD image was shined onto a piece of lens paper or metal, the afterglow
went away:
https://goo.gl/arXYF5
https://goo.gl/cVo2Ev

    The final nail in the coffin to our suspicions was when we then mounted
a plano-convex N-BK7 lens onto the microscope and the effect came back, and
the thicker the lens, the stronger the effect. Also, the effect went away
when we used 540nm light.

With a bit of internet searching I also came across this paper that
confirms there is some visible fluorescence in glass due to trace elements:
http://www.schott.com/d/advanced_optics/87330898-4e56-
4d70-965a-3f03c7bc0c80/1.1/schott_tie-36_fluorescence_of_
optical_glass_us.pdf

Even when I saw the slew, and the first thing that came to mind was
phosphorescence, the last thing that came to mind was that the glass in the
objective itself was the offender, so I wanted to post this to both give
other people a heads-up, and also to see if anyone else has run into this
phenomenon.

Cheers,
   Ben Smith

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Zdenek Svindrych-2]

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*****

Hi Craig,
I'm not that surprised, I've seen luminiscence (in the 0.1 ms range) from
glass coverslips in a two-photon setup. You won't see this effect in a
confocal microscope unless you use strong laser power, high gain, and focus
somewhere into the glass (but be careful, the coverslip chips off easily if
you focus your 2P laser to the glass-water interface :-).


And it's well known that some lenses are better for fluorescence imaging
than others. You won't see the luminiscence decay with a widefield
microscope, the cameras are usually not fast enough. It will just increase
the background in your images. But, to be honest, I would expect this effect
to be extremely weak for modern lenses...

I would definitely double check the LED, too. Many UV LEDs show lot of
luminescence. But if Ben is using DMD to control the illumination (not the
LED itself), this can probably be ruled out, and the DMD itself should be
pretty fast (10 us ?)...

Best, zdenek

--
Zdenek Svindrych, Ph.D.
Research Associate - Imaging Specialist
Department of Biochemistry and Cell Biology
Geisel School of Medicine at Dartmouth

---------- Původní e-mail ----------
Od: Craig Brideau <[hidden email]>
Komu: [hidden email]
Datum: 21. 11. 2017 10:17:12
Předmět: Re: Objective phosphorescence
"*****
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*****

That is quite an unusual finding, Ben. It would be interesting to try a
fused silica lens to see if that gives the same result or not. Glass can
exhibit all sorts of emissions at shorter wavelength but I have never seen
this particular situation. Some LEDs use fluorescence or phosphorescence in
their emission but you seem to have ruled that out. Fused silica *should*
be pure enough to avoid issues at that wavelength.

Craig


On Nov 20, 2017 8:34 PM, "Benjamin E Smith" <[hidden email]>
wrote:

*****
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*****

Hey microscopists,
We observed an odd phenomenon today on a microscope and was wondering if
anyone else has ever seen it. We were using a DMD do a full field flash
with 420nm light during the flyback of the scanning mirror on a 2P imaging
rig. We noticed that after the light was turned off, there was a
millisecond long slewing of the signal that looked a lot like
phosphorescence. In the following image, you can see that the LED is on
for the first portion of the scan, then turns off and the apparent
afterglow: https://goo.gl/2ENHwL

This afterglow was also apparent with an oscilloscope looking at the PMT
and fast mirror signals: https://goo.gl/2AMsvB

We then systematically removed components from the optical path, and
cleaned everything, and we were eventually able to determine that the glass
in the objective itself was glowing, where if the objective was removed and
the DMD image was shined onto a piece of lens paper or metal, the afterglow
went away:
https://goo.gl/arXYF5
https://goo.gl/cVo2Ev

The final nail in the coffin to our suspicions was when we then mounted
a plano-convex N-BK7 lens onto the microscope and the effect came back, and
the thicker the lens, the stronger the effect. Also, the effect went away
when we used 540nm light.

With a bit of internet searching I also came across this paper that
confirms there is some visible fluorescence in glass due to trace elements:
http://www.schott.com/d/advanced_optics/87330898-4e56-
4d70-965a-3f03c7bc0c80/1.1/schott_tie-36_fluorescence_of_
optical_glass_us.pdf

Even when I saw the slew, and the first thing that came to mind was
phosphorescence, the last thing that came to mind was that the glass in the
objective itself was the offender, so I wanted to post this to both give
other people a heads-up, and also to see if anyone else has run into this
phenomenon.

Cheers,
Ben Smith

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA 94720-3200
Tel [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
"

[John Oreopoulos]

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*****

I know of one paper that took a very detailed look at light levels and  
sources of unwanted light in a microscope:

http://www.cell.com/biophysj/abstract/S0006-3495(14)00079-4

"Upon 488-nm excitation with low mW power and EMCCD detection, all  
three tested objectives displayed measurable yellow-green  
autofluorescence (Fig. S2). This instrument fluorescence passes  
undetected as a background offset when imaging the sample-plane. BFP  
imaging with a Bertrand lens allowed us to identify the origin of this  
fluorescence inside the objective."

And take a look at Figure S2.

There likely is some level of fluorescence in all objectives (due to  
the glasses or the adhesives between glasses), but whether it impacts  
your experiment or not probably depends on the relative strength of  
the signal you're trying to detect with set of acquisition parameters  
(laser power, exposure time, probe concentration, etc.)

I suspect there is a lot of variation in this from objective to  
objective, and their likely is room for improvement in terms of  
lowering background signals further, but there is little information  
in the literature about fluorescence/phosphorescence of glasses and  
glass adhesives, and whatever does exist likely resides hidden away in  
the R&D labs of the objective lens makers.

I'm assuming you're identifying the light you're seeing here as  
phosphorescence because of the associated time scales you observe it?

Sincerely,

John Oreopoulos



Quoting Craig Brideau <[hidden email]>:

> *****
> 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.
> *****
>
> That is quite an unusual finding, Ben. It would be interesting to try a
> fused silica lens to see if that gives the same result or not. Glass can
> exhibit all sorts of emissions at shorter wavelength but I have never seen
> this particular situation. Some LEDs use fluorescence or phosphorescence in
> their emission but you seem to have ruled that out. Fused silica *should*
> be pure enough to avoid issues at that wavelength.
>
> Craig
>
>
> On Nov 20, 2017 8:34 PM, "Benjamin E Smith" <[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.
> *****
>
> Hey microscopists,
>    We observed an odd phenomenon today on a microscope and was wondering if
> anyone else has ever seen it.  We were using a DMD do a full field flash
> with 420nm light during the flyback of the scanning mirror on a 2P imaging
> rig.  We noticed that after the light was turned off, there was a
> millisecond long slewing of the signal that looked a lot like
> phosphorescence.  In the following image, you can see that the LED is on
> for the first portion of the scan, then turns off and the apparent
> afterglow: https://goo.gl/2ENHwL
>
> This afterglow was also apparent with an oscilloscope looking at the PMT
> and fast mirror signals: https://goo.gl/2AMsvB
>
>    We then systematically removed components from the optical path, and
> cleaned everything, and we were eventually able to determine that the glass
> in the objective itself was glowing, where if the objective was removed and
> the DMD image was shined onto a piece of lens paper or metal, the afterglow
> went away:
> https://goo.gl/arXYF5
> https://goo.gl/cVo2Ev
>
>     The final nail in the coffin to our suspicions was when we then mounted
> a plano-convex N-BK7 lens onto the microscope and the effect came back, and
> the thicker the lens, the stronger the effect. Also, the effect went away
> when we used 540nm light.
>
> With a bit of internet searching I also came across this paper that
> confirms there is some visible fluorescence in glass due to trace elements:
> http://www.schott.com/d/advanced_optics/87330898-4e56-
> 4d70-965a-3f03c7bc0c80/1.1/schott_tie-36_fluorescence_of_
> optical_glass_us.pdf
>
> Even when I saw the slew, and the first thing that came to mind was
> phosphorescence, the last thing that came to mind was that the glass in the
> objective itself was the offender, so I wanted to post this to both give
> other people a heads-up, and also to see if anyone else has run into this
> phenomenon.
>
> Cheers,
>    Ben Smith
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>

[Craig Brideau]

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

Great points Zdenek. As you say, it seems unusual in a modern objective. I
wonder if there is an internal focus within the lens cluster? Otherwise I'm
trying to figure out how you'd get sufficient energy density within the
glass of the lens for these effects. I suppose if the laser is powerful
enough a focus might not be necessary. Ben also mentioned using a singlet
lens and getting the same result, so perhaps the laser power is high enough
or Ben's particular configuration is sensitive/fast enough?

Craig

On Tue, Nov 21, 2017 at 9:25 AM, <[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 Craig,
> I'm not that surprised, I've seen luminiscence (in the 0.1 ms range) from
> glass coverslips in a two-photon setup. You won't see this effect in a
> confocal microscope unless you use strong laser power, high gain, and focus
> somewhere into the glass (but be careful, the coverslip chips off easily if
> you focus your 2P laser to the glass-water interface :-).
>
>
> And it's well known that some lenses are better for fluorescence imaging
> than others. You won't see the luminiscence decay with a widefield
> microscope, the cameras are usually not fast enough. It will just increase
> the background in your images. But, to be honest, I would expect this
> effect
> to be extremely weak for modern lenses...
>
> I would definitely double check the LED, too. Many UV LEDs show lot of
> luminescence. But if Ben is using DMD to control the illumination (not the
> LED itself), this can probably be ruled out, and the DMD itself should be
> pretty fast (10 us ?)...
>
> Best, zdenek
>
> --
> Zdenek Svindrych, Ph.D.
> Research Associate - Imaging Specialist
> Department of Biochemistry and Cell Biology
> Geisel School of Medicine at Dartmouth
>
> ---------- Původní e-mail ----------
> Od: Craig Brideau <[hidden email]>
> Komu: [hidden email]
> Datum: 21. 11. 2017 10:17:12
> Předmět: Re: Objective phosphorescence
> "*****
> 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.
> *****
>
> That is quite an unusual finding, Ben. It would be interesting to try a
> fused silica lens to see if that gives the same result or not. Glass can
> exhibit all sorts of emissions at shorter wavelength but I have never seen
> this particular situation. Some LEDs use fluorescence or phosphorescence in
> their emission but you seem to have ruled that out. Fused silica *should*
> be pure enough to avoid issues at that wavelength.
>
> Craig
>
>
> On Nov 20, 2017 8:34 PM, "Benjamin E Smith" <[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.
> *****
>
> Hey microscopists,
> We observed an odd phenomenon today on a microscope and was wondering if
> anyone else has ever seen it. We were using a DMD do a full field flash
> with 420nm light during the flyback of the scanning mirror on a 2P imaging
> rig. We noticed that after the light was turned off, there was a
> millisecond long slewing of the signal that looked a lot like
> phosphorescence. In the following image, you can see that the LED is on
> for the first portion of the scan, then turns off and the apparent
> afterglow: https://goo.gl/2ENHwL
>
> This afterglow was also apparent with an oscilloscope looking at the PMT
> and fast mirror signals: https://goo.gl/2AMsvB
>
> We then systematically removed components from the optical path, and
> cleaned everything, and we were eventually able to determine that the glass
> in the objective itself was glowing, where if the objective was removed and
> the DMD image was shined onto a piece of lens paper or metal, the afterglow
> went away:
> https://goo.gl/arXYF5
> https://goo.gl/cVo2Ev
>
> The final nail in the coffin to our suspicions was when we then mounted
> a plano-convex N-BK7 lens onto the microscope and the effect came back, and
> the thicker the lens, the stronger the effect. Also, the effect went away
> when we used 540nm light.
>
> With a bit of internet searching I also came across this paper that
> confirms there is some visible fluorescence in glass due to trace elements:
> http://www.schott.com/d/advanced_optics/87330898-4e56-
> 4d70-965a-3f03c7bc0c80/1.1/schott_tie-36_fluorescence_of_
> optical_glass_us.pdf
>
> Even when I saw the slew, and the first thing that came to mind was
> phosphorescence, the last thing that came to mind was that the glass in the
> objective itself was the offender, so I wanted to post this to both give
> other people a heads-up, and also to see if anyone else has run into this
> phenomenon.
>
> Cheers,
> Ben Smith
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA 94720-3200
> Tel [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
> "
>

[Benjamin Smith]

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

I think the key to seeing this is that we're looking at the after-glow with
non-descanned detectors, and are filling the entire objective with the LED
light (the LED shines at about 100mW onto the back aperture).  The emission
filters block the blue excitation light, and the PMTs are looking at any
light coming out the back aperture, so I think any 2P rig with
non-descanned detectors should be able to see this as long as the LED is
slewed fast enough.  Descanned detectors on an LSCM wouldn't see this, as
the pinhole aperture would block pretty much all of the luminescence coming
from the objective, however if you flashed the 458 nm line of an argon
laser on an LCSM using an AOTF and then looked at the luminescence using a
non-descanned detector on the same rig you should see this (although most
turnkey systems I know of can't be configured to image while the laser is
turned off).

I can also say that the N-BK7 singlets from Thorlabs showed more glow than
the entire beefy 16x/0.8NA Nikon physiology objective, so my guess is that
some glasses such as N-BK7 have this problem more than others such as CaF.

Also, thank you for the paper, John, this is why I love the listerv.  It
definitely looks like they're seeing the same problem.  As for
phosphorescence vs. fluorescence, my wager is that we're seeing both (based
entirely off of the time constants).  I say this because the glow intensity
drops 50-90% (depending on the glass) within a microsecond and then the
rest of the light decays within a millisecond.  I'll leave it to the glass
and objective manufacturers to distinguish triplet states from meta-stable
states, although as you suggested, I don't think they'll be reporting back
anytime soon.

And LED phosphorescence was my first guess at the source.  We had an IR LED
in the condenser path, so my hunch was that the blue light may have been
exciting phosphorescence in the IR LED, but we were able to systematically
narrow it down to glass being the sole source.  Also, it wouldn't be
phosphorescence from the source LED as we're using a DMD to modulate the
light (the LED stays on continuously), and a DMD can flips states in under
a microsecond (if you look at the image in my original post without the
glass in the path, you can see this clean transition).

Finally, judging by the Schott paper, it sounds like this is something can
can not fully removed with most glasses as you will always have some degree
of trace element contamination.  I would be especially interested in
knowing how much this varies batch to batch given how bright our N-BK7
singlets were (i.e. should we start asking for lenses from specific sands
like some sort of wine connoisseur).

Thanks for all of the great feedback,
   Ben Smith




On Tue, Nov 21, 2017 at 2:29 PM, Craig Brideau <[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.
> *****
>
> Great points Zdenek. As you say, it seems unusual in a modern objective. I
> wonder if there is an internal focus within the lens cluster? Otherwise I'm
> trying to figure out how you'd get sufficient energy density within the
> glass of the lens for these effects. I suppose if the laser is powerful
> enough a focus might not be necessary. Ben also mentioned using a singlet
> lens and getting the same result, so perhaps the laser power is high enough
> or Ben's particular configuration is sensitive/fast enough?
>
> Craig
>
> On Tue, Nov 21, 2017 at 9:25 AM, <[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 Craig,
> > I'm not that surprised, I've seen luminiscence (in the 0.1 ms range) from
> > glass coverslips in a two-photon setup. You won't see this effect in a
> > confocal microscope unless you use strong laser power, high gain, and
> focus
> > somewhere into the glass (but be careful, the coverslip chips off easily
> if
> > you focus your 2P laser to the glass-water interface :-).
> >
> >
> > And it's well known that some lenses are better for fluorescence imaging
> > than others. You won't see the luminiscence decay with a widefield
> > microscope, the cameras are usually not fast enough. It will just
> increase
> > the background in your images. But, to be honest, I would expect this
> > effect
> > to be extremely weak for modern lenses...
> >
> > I would definitely double check the LED, too. Many UV LEDs show lot of
> > luminescence. But if Ben is using DMD to control the illumination (not
> the
> > LED itself), this can probably be ruled out, and the DMD itself should be
> > pretty fast (10 us ?)...
> >
> > Best, zdenek
> >
> > --
> > Zdenek Svindrych, Ph.D.
> > Research Associate - Imaging Specialist
> > Department of Biochemistry and Cell Biology
> > Geisel School of Medicine at Dartmouth
> >
> > ---------- Původní e-mail ----------
> > Od: Craig Brideau <[hidden email]>
> > Komu: [hidden email]
> > Datum: 21. 11. 2017 10:17:12
> > Předmět: Re: Objective phosphorescence
> > "*****
> > 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.
> > *****
> >
> > That is quite an unusual finding, Ben. It would be interesting to try a
> > fused silica lens to see if that gives the same result or not. Glass can
> > exhibit all sorts of emissions at shorter wavelength but I have never
> seen
> > this particular situation. Some LEDs use fluorescence or phosphorescence
> in
> > their emission but you seem to have ruled that out. Fused silica *should*
> > be pure enough to avoid issues at that wavelength.
> >
> > Craig
> >
> >
> > On Nov 20, 2017 8:34 PM, "Benjamin E Smith" <[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.
> > *****
> >
> > Hey microscopists,
> > We observed an odd phenomenon today on a microscope and was wondering if
> > anyone else has ever seen it. We were using a DMD do a full field flash
> > with 420nm light during the flyback of the scanning mirror on a 2P
> imaging
> > rig. We noticed that after the light was turned off, there was a
> > millisecond long slewing of the signal that looked a lot like
> > phosphorescence. In the following image, you can see that the LED is on
> > for the first portion of the scan, then turns off and the apparent
> > afterglow: https://goo.gl/2ENHwL
> >
> > This afterglow was also apparent with an oscilloscope looking at the PMT
> > and fast mirror signals: https://goo.gl/2AMsvB
> >
> > We then systematically removed components from the optical path, and
> > cleaned everything, and we were eventually able to determine that the
> glass
> > in the objective itself was glowing, where if the objective was removed
> and
> > the DMD image was shined onto a piece of lens paper or metal, the
> afterglow
> > went away:
> > https://goo.gl/arXYF5
> > https://goo.gl/cVo2Ev
> >
> > The final nail in the coffin to our suspicions was when we then mounted
> > a plano-convex N-BK7 lens onto the microscope and the effect came back,
> and
> > the thicker the lens, the stronger the effect. Also, the effect went away
> > when we used 540nm light.
> >
> > With a bit of internet searching I also came across this paper that
> > confirms there is some visible fluorescence in glass due to trace
> elements:
> > http://www.schott.com/d/advanced_optics/87330898-4e56-
> > 4d70-965a-3f03c7bc0c80/1.1/schott_tie-36_fluorescence_of_
> > optical_glass_us.pdf
> >
> > Even when I saw the slew, and the first thing that came to mind was
> > phosphorescence, the last thing that came to mind was that the glass in
> the
> > objective itself was the offender, so I wanted to post this to both give
> > other people a heads-up, and also to see if anyone else has run into this
> > phenomenon.
> >
> > Cheers,
> > Ben Smith
> >
> > --
> > Benjamin E. Smith, Ph. D.
> > Imaging Specialist, Vision Science
> > University of California, Berkeley
> > 195 Life Sciences Addition
> > Berkeley, CA 94720-3200
> > Tel [phone]
> > Fax [phone]
> > e-mail: [hidden email]
> > http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
> > "
> >
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Martin Wessendorf-2]

*****
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.
*****

Interesting.  I was using the widefield 'scope in my lab today and saw
something that I've seen a thousand times before, but never thought
about: Near-UV excitation causes the optics in my sub-stage condenser to
fluoresce yellow.  However, as others have said, I don't know whether
the source is the glue, the glass, or the housing.

Martin Wessendorf




On 11/20/2017 9:33 PM, Benjamin E Smith 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.
> *****
>
> Hey microscopists,
>     We observed an odd phenomenon today on a microscope and was wondering if
> anyone else has ever seen it.  We were using a DMD do a full field flash
> with 420nm light during the flyback of the scanning mirror on a 2P imaging
> rig.  We noticed that after the light was turned off, there was a
> millisecond long slewing of the signal that looked a lot like
> phosphorescence.  In the following image, you can see that the LED is on
> for the first portion of the scan, then turns off and the apparent
> afterglow: https://goo.gl/2ENHwL
>
> This afterglow was also apparent with an oscilloscope looking at the PMT
> and fast mirror signals: https://goo.gl/2AMsvB
>
>     We then systematically removed components from the optical path, and
> cleaned everything, and we were eventually able to determine that the glass
> in the objective itself was glowing, where if the objective was removed and
> the DMD image was shined onto a piece of lens paper or metal, the afterglow
> went away:
> https://goo.gl/arXYF5
> https://goo.gl/cVo2Ev
>
>      The final nail in the coffin to our suspicions was when we then mounted
> a plano-convex N-BK7 lens onto the microscope and the effect came back, and
> the thicker the lens, the stronger the effect. Also, the effect went away
> when we used 540nm light.
>
> With a bit of internet searching I also came across this paper that
> confirms there is some visible fluorescence in glass due to trace elements:
> http://www.schott.com/d/advanced_optics/87330898-4e56-4d70-965a-3f03c7bc0c80/1.1/schott_tie-36_fluorescence_of_optical_glass_us.pdf
>
> Even when I saw the slew, and the first thing that came to mind was
> phosphorescence, the last thing that came to mind was that the glass in the
> objective itself was the offender, so I wanted to post this to both give
> other people a heads-up, and also to see if anyone else has run into this
> phenomenon.
>
> Cheers,
>     Ben Smith
>

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Gerhard Holst]

*****
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*****

Dear all,

from my experiences with fiber optics and microscopes, I would expect that the additional luminescence might come more from the used glue to fix the lenses within the objectives. I guess for manufacturing reasons, to reduce the times for polymerization often UV hardening might be used. As long as a light beam just passes the lenses, there is only little issue (we are using laser light for widefield illumination of the microscope stage, but we don't have issues with such a background), in case the LED light is able to be spread within the objective casing, some luminescence might excited within the glue and part of it will be guided through the optical system as well.

Could the LED light be better shaped, such that I travels mainly through the lenses?

with best regards,

Gerhard


Dr. Gerhard Holst
Head of Science & Research
[phone]
[phone]

PCO AG, Donaupark 11, 93309 Kelheim, Germany, www.pco.de
USt. ID-Nr. / VAT: DE128590843, Registergericht / Register court: Regensburg HRB 9157
Sitz der Gesellschaft / Registered office: Kelheim, Vorstand / Chairman: Dr. Emil Ott
Vorsitzender des Aufsichtsrats / Chairman of the supervisory board: Johann Plöb


-----Ursprüngliche Nachricht-----
Von: Confocal Microscopy List [mailto:[hidden email]] Im Auftrag von Martin Wessendorf
Gesendet: Mittwoch, 22. November 2017 00:16
An: [hidden email]
Betreff: Re: Objective phosphorescence

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Interesting.  I was using the widefield 'scope in my lab today and saw something that I've seen a thousand times before, but never thought
about: Near-UV excitation causes the optics in my sub-stage condenser to fluoresce yellow.  However, as others have said, I don't know whether the source is the glue, the glass, or the housing.

Martin Wessendorf




On 11/20/2017 9:33 PM, Benjamin E Smith 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.
> *****
>
> Hey microscopists,
>     We observed an odd phenomenon today on a microscope and was
> wondering if anyone else has ever seen it.  We were using a DMD do a
> full field flash with 420nm light during the flyback of the scanning
> mirror on a 2P imaging rig.  We noticed that after the light was
> turned off, there was a millisecond long slewing of the signal that
> looked a lot like phosphorescence.  In the following image, you can
> see that the LED is on for the first portion of the scan, then turns
> off and the apparent
> afterglow: https://goo.gl/2ENHwL
>
> This afterglow was also apparent with an oscilloscope looking at the
> PMT and fast mirror signals: https://goo.gl/2AMsvB
>
>     We then systematically removed components from the optical path,
> and cleaned everything, and we were eventually able to determine that
> the glass in the objective itself was glowing, where if the objective
> was removed and the DMD image was shined onto a piece of lens paper or
> metal, the afterglow went away:
> https://goo.gl/arXYF5
> https://goo.gl/cVo2Ev
>
>      The final nail in the coffin to our suspicions was when we then
> mounted a plano-convex N-BK7 lens onto the microscope and the effect
> came back, and the thicker the lens, the stronger the effect. Also,
> the effect went away when we used 540nm light.
>
> With a bit of internet searching I also came across this paper that
> confirms there is some visible fluorescence in glass due to trace elements:
> http://www.schott.com/d/advanced_optics/87330898-4e56-4d70-965a-3f03c7
> bc0c80/1.1/schott_tie-36_fluorescence_of_optical_glass_us.pdf
>
> Even when I saw the slew, and the first thing that came to mind was
> phosphorescence, the last thing that came to mind was that the glass
> in the objective itself was the offender, so I wanted to post this to
> both give other people a heads-up, and also to see if anyone else has
> run into this phenomenon.
>
> Cheers,
>     Ben Smith
>

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Benjamin Smith]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

On thing to note about the glue hypothesis is that the singlet lens (i.e.
no glue) showed the brightest luminescence.  Also, the glass in optical
fibers is a whole different beast, where the glass is actually deposited
via very high purity gasses: https://www.youtube.com/watch?v=u1DRrAhQJtM

One of my favorite facts about the glass in fibers is that if the ocean was
as clear as an optical fiber, you could clearly see the bottom.   As was
suggested by Craig, fused silica lenses should be of a similar purity, so
they should show a similar low level of luminescence to optical fibers.  If
I had one floating around I'd try it, but we normally work in the nIR as
opposed to UV.

-Ben Smith

On Tue, Nov 21, 2017 at 11:25 PM, Gerhard Holst <[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 all,
>
> from my experiences with fiber optics and microscopes, I would expect that
> the additional luminescence might come more from the used glue to fix the
> lenses within the objectives. I guess for manufacturing reasons, to reduce
> the times for polymerization often UV hardening might be used. As long as a
> light beam just passes the lenses, there is only little issue (we are using
> laser light for widefield illumination of the microscope stage, but we
> don't have issues with such a background), in case the LED light is able to
> be spread within the objective casing, some luminescence might excited
> within the glue and part of it will be guided through the optical system as
> well.
>
> Could the LED light be better shaped, such that I travels mainly through
> the lenses?
>
> with best regards,
>
> Gerhard
>
>
> Dr. Gerhard Holst
> Head of Science & Research
> [phone]
> [phone]
>
> PCO AG, Donaupark 11, 93309 Kelheim, Germany, www.pco.de
> USt. ID-Nr. / VAT: DE128590843, Registergericht / Register court:
> Regensburg HRB 9157
> Sitz der Gesellschaft / Registered office: Kelheim, Vorstand / Chairman:
> Dr. Emil Ott
> Vorsitzender des Aufsichtsrats / Chairman of the supervisory board: Johann
> Plöb
>
>
> -----Ursprüngliche Nachricht-----
> Von: Confocal Microscopy List [mailto:[hidden email]]
> Im Auftrag von Martin Wessendorf
> Gesendet: Mittwoch, 22. November 2017 00:16
> An: [hidden email]
> Betreff: Re: Objective phosphorescence
>
> *****
> 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.
> *****
>
> Interesting.  I was using the widefield 'scope in my lab today and saw
> something that I've seen a thousand times before, but never thought
> about: Near-UV excitation causes the optics in my sub-stage condenser to
> fluoresce yellow.  However, as others have said, I don't know whether the
> source is the glue, the glass, or the housing.
>
> Martin Wessendorf
>
>
>
>
> On 11/20/2017 9:33 PM, Benjamin E Smith 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.
> > *****
> >
> > Hey microscopists,
> >     We observed an odd phenomenon today on a microscope and was
> > wondering if anyone else has ever seen it.  We were using a DMD do a
> > full field flash with 420nm light during the flyback of the scanning
> > mirror on a 2P imaging rig.  We noticed that after the light was
> > turned off, there was a millisecond long slewing of the signal that
> > looked a lot like phosphorescence.  In the following image, you can
> > see that the LED is on for the first portion of the scan, then turns
> > off and the apparent
> > afterglow: https://goo.gl/2ENHwL
> >
> > This afterglow was also apparent with an oscilloscope looking at the
> > PMT and fast mirror signals: https://goo.gl/2AMsvB
> >
> >     We then systematically removed components from the optical path,
> > and cleaned everything, and we were eventually able to determine that
> > the glass in the objective itself was glowing, where if the objective
> > was removed and the DMD image was shined onto a piece of lens paper or
> > metal, the afterglow went away:
> > https://goo.gl/arXYF5
> > https://goo.gl/cVo2Ev
> >
> >      The final nail in the coffin to our suspicions was when we then
> > mounted a plano-convex N-BK7 lens onto the microscope and the effect
> > came back, and the thicker the lens, the stronger the effect. Also,
> > the effect went away when we used 540nm light.
> >
> > With a bit of internet searching I also came across this paper that
> > confirms there is some visible fluorescence in glass due to trace
> elements:
> > http://www.schott.com/d/advanced_optics/87330898-4e56-4d70-965a-3f03c7
> > bc0c80/1.1/schott_tie-36_fluorescence_of_optical_glass_us.pdf
> >
> > Even when I saw the slew, and the first thing that came to mind was
> > phosphorescence, the last thing that came to mind was that the glass
> > in the objective itself was the offender, so I wanted to post this to
> > both give other people a heads-up, and also to see if anyone else has
> > run into this phenomenon.
> >
> > Cheers,
> >     Ben Smith
> >
>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]
>

--
Benjamin E. Smith, Ph. D.
Imaging Specialist, Vision Science
University of California, Berkeley
195 Life Sciences Addition
Berkeley, CA  94720-3200
Tel  [phone]
Fax [phone]
e-mail: [hidden email]
http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>

[Craig Brideau]

*****
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.
*****

My first instinct was to blame the glue as well, but when Ben mentioned
testing with a singlet I realized it was really the glass. That said, many
optical epoxies are UV cured, so they absorb more at the bluer wavelengths.
I don't know how this would translate into fluorescence or phosphorescence
but you would definitely see more absorption if a lens made liberal use of
such glue in its construction.

Craig

On Wed, Nov 22, 2017 at 1:17 AM, Benjamin E Smith <
[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.
> *****
>
> On thing to note about the glue hypothesis is that the singlet lens (i.e.
> no glue) showed the brightest luminescence.  Also, the glass in optical
> fibers is a whole different beast, where the glass is actually deposited
> via very high purity gasses: https://www.youtube.com/watch?v=u1DRrAhQJtM
>
> One of my favorite facts about the glass in fibers is that if the ocean was
> as clear as an optical fiber, you could clearly see the bottom.   As was
> suggested by Craig, fused silica lenses should be of a similar purity, so
> they should show a similar low level of luminescence to optical fibers.  If
> I had one floating around I'd try it, but we normally work in the nIR as
> opposed to UV.
>
> -Ben Smith
>
> On Tue, Nov 21, 2017 at 11:25 PM, Gerhard Holst <[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 all,
> >
> > from my experiences with fiber optics and microscopes, I would expect
> that
> > the additional luminescence might come more from the used glue to fix the
> > lenses within the objectives. I guess for manufacturing reasons, to
> reduce
> > the times for polymerization often UV hardening might be used. As long
> as a
> > light beam just passes the lenses, there is only little issue (we are
> using
> > laser light for widefield illumination of the microscope stage, but we
> > don't have issues with such a background), in case the LED light is able
> to
> > be spread within the objective casing, some luminescence might excited
> > within the glue and part of it will be guided through the optical system
> as
> > well.
> >
> > Could the LED light be better shaped, such that I travels mainly through
> > the lenses?
> >
> > with best regards,
> >
> > Gerhard
> >
> >
> > Dr. Gerhard Holst
> > Head of Science & Research
> > [phone]
> > [phone]
> >
> > PCO AG, Donaupark 11, 93309 Kelheim, Germany, www.pco.de
> > USt. ID-Nr. / VAT: DE128590843, Registergericht / Register court:
> > Regensburg HRB 9157
> > Sitz der Gesellschaft / Registered office: Kelheim, Vorstand / Chairman:
> > Dr. Emil Ott
> > Vorsitzender des Aufsichtsrats / Chairman of the supervisory board:
> Johann
> > Plöb
> >
> >
> > -----Ursprüngliche Nachricht-----
> > Von: Confocal Microscopy List [mailto:[hidden email]]
> > Im Auftrag von Martin Wessendorf
> > Gesendet: Mittwoch, 22. November 2017 00:16
> > An: [hidden email]
> > Betreff: Re: Objective phosphorescence
> >
> > *****
> > 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.
> > *****
> >
> > Interesting.  I was using the widefield 'scope in my lab today and saw
> > something that I've seen a thousand times before, but never thought
> > about: Near-UV excitation causes the optics in my sub-stage condenser to
> > fluoresce yellow.  However, as others have said, I don't know whether the
> > source is the glue, the glass, or the housing.
> >
> > Martin Wessendorf
> >
> >
> >
> >
> > On 11/20/2017 9:33 PM, Benjamin E Smith 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.
> > > *****
> > >
> > > Hey microscopists,
> > >     We observed an odd phenomenon today on a microscope and was
> > > wondering if anyone else has ever seen it.  We were using a DMD do a
> > > full field flash with 420nm light during the flyback of the scanning
> > > mirror on a 2P imaging rig.  We noticed that after the light was
> > > turned off, there was a millisecond long slewing of the signal that
> > > looked a lot like phosphorescence.  In the following image, you can
> > > see that the LED is on for the first portion of the scan, then turns
> > > off and the apparent
> > > afterglow: https://goo.gl/2ENHwL
> > >
> > > This afterglow was also apparent with an oscilloscope looking at the
> > > PMT and fast mirror signals: https://goo.gl/2AMsvB
> > >
> > >     We then systematically removed components from the optical path,
> > > and cleaned everything, and we were eventually able to determine that
> > > the glass in the objective itself was glowing, where if the objective
> > > was removed and the DMD image was shined onto a piece of lens paper or
> > > metal, the afterglow went away:
> > > https://goo.gl/arXYF5
> > > https://goo.gl/cVo2Ev
> > >
> > >      The final nail in the coffin to our suspicions was when we then
> > > mounted a plano-convex N-BK7 lens onto the microscope and the effect
> > > came back, and the thicker the lens, the stronger the effect. Also,
> > > the effect went away when we used 540nm light.
> > >
> > > With a bit of internet searching I also came across this paper that
> > > confirms there is some visible fluorescence in glass due to trace
> > elements:
> > > http://www.schott.com/d/advanced_optics/87330898-4e56-4d70-965a-3f03c7
> > > bc0c80/1.1/schott_tie-36_fluorescence_of_optical_glass_us.pdf
> > >
> > > Even when I saw the slew, and the first thing that came to mind was
> > > phosphorescence, the last thing that came to mind was that the glass
> > > in the objective itself was the offender, so I wanted to post this to
> > > both give other people a heads-up, and also to see if anyone else has
> > > run into this phenomenon.
> > >
> > > Cheers,
> > >     Ben Smith
> > >
> >
> > --
> > Martin Wessendorf, Ph.D.                   office: [phone]
> > Assoc Prof, Dept Neuroscience                 lab: [phone]
> > University of Minnesota             Preferred FAX: [phone]
> > 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> > Minneapolis, MN  55455                    e-mail: [hidden email]
> >
>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>

[Oshel, Philip Eugene]

*****
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.
*****

Is it the glass or the coating?

Phil
-------------
Philip Oshel    
Imaging Facility Director
Biology Department
1304 Biosciences
1455 Calumet Ct.
Central Michigan University
Mt. Pleasant, MI 48859
989 774-3576 office
989 774-7567 lab









-----Original Message-----
From: Confocal Microscopy List <[hidden email]> on behalf of Craig Brideau <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Wednesday,  22November, 2017 at 12:09
To: "[hidden email]" <[hidden email]>
Subject: Re: Objective phosphorescence

*****
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.
*****

My first instinct was to blame the glue as well, but when Ben mentioned
testing with a singlet I realized it was really the glass. That said, many
optical epoxies are UV cured, so they absorb more at the bluer wavelengths.
I don't know how this would translate into fluorescence or phosphorescence
but you would definitely see more absorption if a lens made liberal use of
such glue in its construction.

Craig

On Wed, Nov 22, 2017 at 1:17 AM, Benjamin E Smith <
[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.
> *****
>
> On thing to note about the glue hypothesis is that the singlet lens (i.e.
> no glue) showed the brightest luminescence.  Also, the glass in optical
> fibers is a whole different beast, where the glass is actually deposited
> via very high purity gasses: https://www.youtube.com/watch?v=u1DRrAhQJtM
>
> One of my favorite facts about the glass in fibers is that if the ocean was
> as clear as an optical fiber, you could clearly see the bottom.   As was
> suggested by Craig, fused silica lenses should be of a similar purity, so
> they should show a similar low level of luminescence to optical fibers.  If
> I had one floating around I'd try it, but we normally work in the nIR as
> opposed to UV.
>
> -Ben Smith
>
> On Tue, Nov 21, 2017 at 11:25 PM, Gerhard Holst <[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 all,
> >
> > from my experiences with fiber optics and microscopes, I would expect
> that
> > the additional luminescence might come more from the used glue to fix the
> > lenses within the objectives. I guess for manufacturing reasons, to
> reduce
> > the times for polymerization often UV hardening might be used. As long
> as a
> > light beam just passes the lenses, there is only little issue (we are
> using
> > laser light for widefield illumination of the microscope stage, but we
> > don't have issues with such a background), in case the LED light is able
> to
> > be spread within the objective casing, some luminescence might excited
> > within the glue and part of it will be guided through the optical system
> as
> > well.
> >
> > Could the LED light be better shaped, such that I travels mainly through
> > the lenses?
> >
> > with best regards,
> >
> > Gerhard
> >
> >
> > Dr. Gerhard Holst
> > Head of Science & Research
> > [phone]
> > [phone]
> >
> > PCO AG, Donaupark 11, 93309 Kelheim, Germany, www.pco.de
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> > Dr. Emil Ott
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> Johann
> > Plöb
> >
> >
> > -----Ursprüngliche Nachricht-----
> > Von: Confocal Microscopy List [mailto:[hidden email]]
> > Im Auftrag von Martin Wessendorf
> > Gesendet: Mittwoch, 22. November 2017 00:16
> > An: [hidden email]
> > Betreff: Re: Objective phosphorescence
> >
> > *****
> > 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.
> > *****
> >
> > Interesting.  I was using the widefield 'scope in my lab today and saw
> > something that I've seen a thousand times before, but never thought
> > about: Near-UV excitation causes the optics in my sub-stage condenser to
> > fluoresce yellow.  However, as others have said, I don't know whether the
> > source is the glue, the glass, or the housing.
> >
> > Martin Wessendorf
> >
> >
> >
> >
> > On 11/20/2017 9:33 PM, Benjamin E Smith 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.
> > > *****
> > >
> > > Hey microscopists,
> > >     We observed an odd phenomenon today on a microscope and was
> > > wondering if anyone else has ever seen it.  We were using a DMD do a
> > > full field flash with 420nm light during the flyback of the scanning
> > > mirror on a 2P imaging rig.  We noticed that after the light was
> > > turned off, there was a millisecond long slewing of the signal that
> > > looked a lot like phosphorescence.  In the following image, you can
> > > see that the LED is on for the first portion of the scan, then turns
> > > off and the apparent
> > > afterglow: https://goo.gl/2ENHwL
> > >
> > > This afterglow was also apparent with an oscilloscope looking at the
> > > PMT and fast mirror signals: https://goo.gl/2AMsvB
> > >
> > >     We then systematically removed components from the optical path,
> > > and cleaned everything, and we were eventually able to determine that
> > > the glass in the objective itself was glowing, where if the objective
> > > was removed and the DMD image was shined onto a piece of lens paper or
> > > metal, the afterglow went away:
> > > https://goo.gl/arXYF5
> > > https://goo.gl/cVo2Ev
> > >
> > >      The final nail in the coffin to our suspicions was when we then
> > > mounted a plano-convex N-BK7 lens onto the microscope and the effect
> > > came back, and the thicker the lens, the stronger the effect. Also,
> > > the effect went away when we used 540nm light.
> > >
> > > With a bit of internet searching I also came across this paper that
> > > confirms there is some visible fluorescence in glass due to trace
> > elements:
> > > http://www.schott.com/d/advanced_optics/87330898-4e56-4d70-965a-3f03c7
> > > bc0c80/1.1/schott_tie-36_fluorescence_of_optical_glass_us.pdf
> > >
> > > Even when I saw the slew, and the first thing that came to mind was
> > > phosphorescence, the last thing that came to mind was that the glass
> > > in the objective itself was the offender, so I wanted to post this to
> > > both give other people a heads-up, and also to see if anyone else has
> > > run into this phenomenon.
> > >
> > > Cheers,
> > >     Ben Smith
> > >
> >
> > --
> > Martin Wessendorf, Ph.D.                   office: [phone]
> > Assoc Prof, Dept Neuroscience                 lab: [phone]
> > University of Minnesota             Preferred FAX: [phone]
> > 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> > Minneapolis, MN  55455                    e-mail: [hidden email]
> >
>
>
>
> --
> Benjamin E. Smith, Ph. D.
> Imaging Specialist, Vision Science
> University of California, Berkeley
> 195 Life Sciences Addition
> Berkeley, CA  94720-3200
> Tel  [phone]
> Fax [phone]
> e-mail: [hidden email]
> http://vision.berkeley.edu/?page_id=5635 <http://vision.berkeley.edu/>
>