PMT damage from too much light?

[ChrisWilms]

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Hi all,

I've recently started working on an older 2-photon LSM (Prairie  
Ultima) using Hamamatsu R3896 PMTs. Compared to what I've seen on  
comparable systems in the past the images are very noisy. Given that  
the system has been used by many people over the years and was  
probably not always treated as well as it should, I have the suspicion  
that the PMTs might be degraded after having "seen" too much incident  
light.

 From my understanding, this should cause a loss in signal. Would it  
also explain the increase in noise?

If so, how would I go about verifying if this the case?

Thankful for any help,

Christian

[James Pawley]

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>*****
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>
>Hi all,
>
>I've recently started working on an older 2-photon LSM (Prairie
>Ultima) using Hamamatsu R3896 PMTs. Compared to what I've seen on
>comparable systems in the past the images are very noisy. Given that
>the system has been used by many people over the years and was
>probably not always treated as well as it should, I have the
>suspicion that the PMTs might be degraded after having "seen" too
>much incident light.
>
>From my understanding, this should cause a loss in signal. Would it
>also explain the increase in noise?
>
>If so, how would I go about verifying if this the case?
>
>Thankful for any help,
>
>Christian

Hi Christian,

25mm diam PMTs that are completely shielded from any sources of light
(and at room temp) should not see more than 30k noise pulses/second.
Assuming you can 512x512, you might expect to see at most one noise
pulse in every tenth pixel. PMTs with smaller photocathode areas
should be proportionally less.

My thought, however, was more along the lines of stray light. With
wide-field collection you have to be  absolutely sure that room light
(even from the LEDs on some equipment in an otherwise dark room) is
not entering the objective or reaching your PMTs through gaps in the
light guides. Is there some what you can wrap the PMT in black tape
just for comparison?

JP
--
***************************************************************************
Prof. James B. Pawley,                          Ph.
608-238-3953                        
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/                  Application
deadline 3/16/2012
               "If it ain't diffraction, it must be statistics." Anon.

[Guy Cox-2]

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But, surely, if a PMT has lost sensitivity the signal to noise must automatically get worse.

                                         Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone [phone]     Fax [phone]
             Mobile [phone]
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of James Pawley
Sent: Thursday, 19 January 2012 4:19 AM
To: [hidden email]
Subject: Re: PMT damage from too much light?

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>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi all,
>
>I've recently started working on an older 2-photon LSM (Prairie
>Ultima) using Hamamatsu R3896 PMTs. Compared to what I've seen on
>comparable systems in the past the images are very noisy. Given that
>the system has been used by many people over the years and was
>probably not always treated as well as it should, I have the
>suspicion that the PMTs might be degraded after having "seen" too
>much incident light.
>
>From my understanding, this should cause a loss in signal. Would it
>also explain the increase in noise?
>
>If so, how would I go about verifying if this the case?
>
>Thankful for any help,
>
>Christian

Hi Christian,

25mm diam PMTs that are completely shielded from any sources of light
(and at room temp) should not see more than 30k noise pulses/second.
Assuming you can 512x512, you might expect to see at most one noise
pulse in every tenth pixel. PMTs with smaller photocathode areas
should be proportionally less.

My thought, however, was more along the lines of stray light. With
wide-field collection you have to be  absolutely sure that room light
(even from the LEDs on some equipment in an otherwise dark room) is
not entering the objective or reaching your PMTs through gaps in the
light guides. Is there some what you can wrap the PMT in black tape
just for comparison?

JP
--
***************************************************************************
Prof. James B. Pawley,                          Ph.
608-238-3953                        
21. N. Prospect Ave. Madison, WI 53726 USA
[hidden email]
3D Microscopy of Living Cells Course, June 9-21, 2012, UBC, Vancouver Canada
Info: http://www.3dcourse.ubc.ca/                  Application
deadline 3/16/2012
               "If it ain't diffraction, it must be statistics." Anon.

[ChrisWilms]

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Hi James and Guy,

thanks for the quick and helpful responses.

I will test the dark counts of the PMTs for starters and also see if I  
might be picking up stray light. It is an obvious thing to do that I  
hadn't explicitly checked for yet.

And of course a loss of sensitivity would lead to a poorer SNR. My  
impression is that that dark counts are way up as well. I'll know more  
in a bit.

Best, Christian


> But, surely, if a PMT has lost sensitivity the signal to noise must  
> automatically get worse.
>
>                                         Guy

>> Hi Christian,
>>
>> 25mm diam PMTs that are completely shielded from any sources of light
>> (and at room temp) should not see more than 30k noise pulses/second.
>> Assuming you can 512x512, you might expect to see at most one noise
>> pulse in every tenth pixel. PMTs with smaller photocathode areas
>> should be proportionally less.
>>
>> My thought, however, was more along the lines of stray light. With
>> wide-field collection you have to be  absolutely sure that room light
>> (even from the LEDs on some equipment in an otherwise dark room) is
>> not entering the objective or reaching your PMTs through gaps in the
>> light guides. Is there some what you can wrap the PMT in black tape
>> just for comparison?
>>
>> JP

[ChrisWilms]

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I just realized, I had nearly forgotten to follow up on this. Yes: it  
turned out I had a source of stray light in the dark box surrounding  
the scope: the glow in the dark tags used to label the filter wheel  
positions on the original Olympus body. Removing them gave me a major  
improvement, blackening several polished (and thus reflecting)  
surfaces in the detection path did the rest.

Just in case someone stumbles over similar problems.

Thanks for all helpful hints,

Christian

> My thought, however, was more along the lines of stray light. With
> wide-field collection you have to be  absolutely sure that room light
> (even from the LEDs on some equipment in an otherwise dark room) is
> not entering the objective or reaching your PMTs through gaps in the
> light guides. Is there some what you can wrap the PMT in black tape
> just for comparison?