Re: Objective phosphorescence

Posted by John Oreopoulos on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Objective-phosphorescence-tp7587588p7587595.html

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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]>:

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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]
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>