Re: Objective phosphorescence

Posted by Zdenek Svindrych-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Objective-phosphorescence-tp7587588p7587594.html

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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
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Fax [phone]
e-mail: [hidden email]
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