Re: Objective phosphorescence

Posted by Benjamin Smith on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Objective-phosphorescence-tp7587588p7587602.html

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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:

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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 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/>