2-P of red fluorescent proteins

[Boswell, Carl A - (cboswell)]

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Hi all,
Does anyone have experience with seeing DSRed or mCherry using a Ti:Sa 2-P
system?  If it doesn't work, what are the alternatives?

Thanks,
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

[Graham Ellis-Davies]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Carl,
Svoboda published a paper in PLOS Biology in 2006 vol. 4 (11) pp. e370, using mCherry.

Carl Boswell <[hidden email]> wrote:

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Hi all,
Does anyone have experience with seeing DSRed or mCherry using a Ti:Sa 2-P
system? If it doesn't work, what are the alternatives?

Thanks,
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

Graham CR Ellis-Davies, PhD.
Associate Professor
Department of Pharmacology & Physiology
Drexel University College of Medicine
Philadelphia
PA 19102
USA

T 215 762 8794
E [hidden email] or [hidden email]
www.pages.drexel.edu/~ge24
cagedcalcium.blogspot.com

---

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[Haberman, Ann]

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I've heard good things about td-tomato, and am looking forward to
trying it. My limited experience with mRFP has been frustrating as it
seems to require a very long wavelength (>950 nm) at which most
Ti:Saph don't typically have that much power.
-Ann

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi all,
>Does anyone have experience with seeing DSRed or mCherry using a
>Ti:Sa 2-P system?  If it doesn't work, what are the alternatives?
>
>Thanks,
>Carl
>
>Carl A. Boswell, Ph.D.
>Molecular and Cellular Biology
>University of Arizona
>520-954-7053
>FAX [phone]

--

Ann Haberman, PhD
Department of Laboratory Medicine
Yale University School of Medicine
1 Gilbert  St.
TAC S541
New Haven, CT 06510

203-785-7349
203-785-5415 (fax)
[hidden email]

[Quoc Thang Nguyen]

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        Dear all,

I have encountered a good deal of folklore regarding PMT operation in
scanning microscopy. Maybe someone in the list can confirm or debunk
these informations.

1) Some insist that PMTs should never be turned on and off during an
experiment, but instead that their voltage should be turned way down
when not in use. Is that true?

2) We have routinely exposed accidentally our PMTs to ambient light
(sometime for a few minutes at time). The dark noise should have
increased. Is this irreversible? Does sensitivity take a hit as well?
We use Hamamatsu PMTs with multialkali (R6357) or GaAs  photocathodes
(H7422-40).

3) Some microscope vendors claim that they hand-pick their PMTs from a
large lot. Does this make a difference?

4) What is the advantage of changing the PMT voltage except for
providing a crude gain control? Would it be better to always set the
voltage to an optimal value for best signal to noise ratio and amplify
the output current?

5) Why is photon counting not widely available on commercial scopes,
since computers would deal easily with digitized counts from the PMT?

Thanks to all.


Quoc Thang NGUYEN, Ph.D.
Assistant Project Scientist
Physics Department, UCSD
e-mail: [hidden email]
Cell Phone: [phone]
Fax: [phone]

[mmodel]

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I only have an opinion on your question #4. There are a few important
differences between the amplifier gain and the voltage. 1) With voltage
you can vary sensitivity in a much wider range, since sensitivity
depends on V nonlinearly (as a power function) 2) Voltage doesn't affect
the background, the gain does. 3) Not all confocal systems allow gain
control (I think Leica doesn't, at least some models). 4) It seems to me
that S/N gets much worse with voltage increase than with the gain
increase.

Michael Model, Ph.D.
Confocal Microscopy Core
Dpt. Biological Sciences
Kent State University
Kent, OH 44242
tel. [phone]

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Quoc Thang Nguyen
Sent: Thursday, March 13, 2008 1:23 PM
To: [hidden email]
Subject: PMT folklore

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

        Dear all,

I have encountered a good deal of folklore regarding PMT operation in
scanning microscopy. Maybe someone in the list can confirm or debunk
these informations.

1) Some insist that PMTs should never be turned on and off during an
experiment, but instead that their voltage should be turned way down
when not in use. Is that true?

2) We have routinely exposed accidentally our PMTs to ambient light
(sometime for a few minutes at time). The dark noise should have
increased. Is this irreversible? Does sensitivity take a hit as well?
We use Hamamatsu PMTs with multialkali (R6357) or GaAs  photocathodes
(H7422-40).

3) Some microscope vendors claim that they hand-pick their PMTs from a
large lot. Does this make a difference?

4) What is the advantage of changing the PMT voltage except for
providing a crude gain control? Would it be better to always set the
voltage to an optimal value for best signal to noise ratio and amplify
the output current?

5) Why is photon counting not widely available on commercial scopes,
since computers would deal easily with digitized counts from the PMT?

Thanks to all.


Quoc Thang NGUYEN, Ph.D.
Assistant Project Scientist
Physics Department, UCSD
e-mail: [hidden email]
Cell Phone: [phone]
Fax: [phone]

[Jeremy Adler]

Search the CONFOCAL archive at
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re point 5    Photon counting.
BioRad offered photon counting.
 
We actually want a photon count - it is far more useful than the integrated PMT output and gives a very good indication of the image quality.
An estimate, based on the PMT output divided by the average PMT response to a single photon hit, is going to be less accurate since there is variability in the PMT response.  
This is obviously more significant when the counts are low - in normal confocal microscopy.
 
 
Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden

________________________________

From: Confocal Microscopy List on behalf of Quoc Thang Nguyen
Sent: Thu 3/13/2008 18:22
To: [hidden email]
Subject: PMT folklore



Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

        Dear all,

I have encountered a good deal of folklore regarding PMT operation in
scanning microscopy. Maybe someone in the list can confirm or debunk
these informations.

1) Some insist that PMTs should never be turned on and off during an
experiment, but instead that their voltage should be turned way down
when not in use. Is that true?

2) We have routinely exposed accidentally our PMTs to ambient light
(sometime for a few minutes at time). The dark noise should have
increased. Is this irreversible? Does sensitivity take a hit as well?
We use Hamamatsu PMTs with multialkali (R6357) or GaAs  photocathodes
(H7422-40).

3) Some microscope vendors claim that they hand-pick their PMTs from a
large lot. Does this make a difference?

4) What is the advantage of changing the PMT voltage except for
providing a crude gain control? Would it be better to always set the
voltage to an optimal value for best signal to noise ratio and amplify
the output current?

5) Why is photon counting not widely available on commercial scopes,
since computers would deal easily with digitized counts from the PMT?

Thanks to all.


Quoc Thang NGUYEN, Ph.D.
Assistant Project Scientist
Physics Department, UCSD
e-mail: [hidden email]
Cell Phone: [phone]
Fax: [phone]

[Michael Weber-4]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

1) This is afaik the way it's done in commercial confocals. PMTs need some
time to stabilize and they also heat up over time, so I doubt that they
can be triggered.

3) I think selection at some point in the production chain makes sense;
PMTs differ quite a lot (no numbers here, unfortunately), but I am sure
also Hamamatsu selects them before sending them to vendors. A commercial
response would be interesting here!

4) Good question, I am also interested in that one. As already mentioned,
voltage offers a wider range, but at a certain level S/N gets down, here
one could think about the gain instead. But every time I used it in the
past (Zeiss "Amplifier Gain", Olympus "Gain") I was quite disappointed...
and Leica doesn't have "Gain" at all.

5) Olympus and Zeiss have it for PMTs, and it's also available in FCS
upgrades with APDs. Older PMT systems where maybe to noisy for this?!

Michael

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear all,
>
> I have encountered a good deal of folklore regarding PMT operation in
> scanning microscopy. Maybe someone in the list can confirm or debunk
> these informations.
>
> 1) Some insist that PMTs should never be turned on and off during an
> experiment, but instead that their voltage should be turned way down
> when not in use. Is that true?
>
> 2) We have routinely exposed accidentally our PMTs to ambient light
> (sometime for a few minutes at time). The dark noise should have
> increased. Is this irreversible? Does sensitivity take a hit as well?
> We use Hamamatsu PMTs with multialkali (R6357) or GaAs  photocathodes
> (H7422-40).
>
> 3) Some microscope vendors claim that they hand-pick their PMTs from a
> large lot. Does this make a difference?
>
> 4) What is the advantage of changing the PMT voltage except for
> providing a crude gain control? Would it be better to always set the
> voltage to an optimal value for best signal to noise ratio and amplify
> the output current?
>
> 5) Why is photon counting not widely available on commercial scopes,
> since computers would deal easily with digitized counts from the PMT?
>
> Thanks to all.
>
>
> Quoc Thang NGUYEN, Ph.D.
> Assistant Project Scientist
> Physics Department, UCSD
> e-mail: [hidden email]
> Cell Phone: [phone]
> Fax: [phone]

[Dale Callaham]

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Hi All,

There is some very good technical reference material on PMTs in the
Hamamatsu PMT Handbook at their website:
http://sales.hamamatsu.com/assets/pdf/catsandguides/PMT_handbook_v3aE.pdf
And a similar one from ElectronTubes (formerly Thorne_EMI):
http://www.electrontubes.com/info/wedo.html
These are free downloads and have a wealth of info to dispel "folklore"....


Regarding:

2) It depends on the design of the HV circuit and if it has limiting. A
stiff HV supply with no limiting will allow an enormous current to reach
the anode if exposed to bright light. The dynode materials are various
materials that, as I understand it, can be evaporated to deposit inside
the PMT envelope, this reducing the sensitivity (as the dynode secondary
emitter materials are depleted) and give more leakage currents. Having
said this, no unit should ever be supplied with unrestricted voltage
this way. The normal full scale signal output currents are very small
and the circuit should have some limits that do not restrict linearity.
The instrument with the PMT can also use a secondary light sensor to
shut off the PMT HV if it is exposed to ambient light. In the SEms that
I am familiar with the PMT HV is derived from the SEM HV generation, and
so it is shut off if the chamber is vented.

3) Selection definitely makes sense for critical applications. These are
individual devices with a spread in the characteristics and if some
feature (low counts/sec, tight pulse shape, etc.) really matters to you,
a "good" one can be selected from the production run.

4) The operating voltage. It is an easy way to change gain for general
purpose detectors. Although the amplification is non-linear with
Voltage, for a given voltage it is linear. The electron multiplier is
extremely low noise amplification, at it's best in photon counting, but
lower noise than most electronics of similar bandwidth. Read some of the
Hamamatsu lit on photon counting for a specific case of finding the
optimum operation voltage (for photon counting) of a particular tube.

5) Photon counting tubes are a different design in general and require
very different preamps and discriminators/window discriminators.
Although Biorad offered a photon counting setting, I am not convinced it
was very good at all. The PMTs they used were NOT considered to be good
counting tubes.

Dale Callaham


Michael Weber wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 1) This is afaik the way it's done in commercial confocals. PMTs need some
> time to stabilize and they also heat up over time, so I doubt that they
> can be triggered.
>
> 3) I think selection at some point in the production chain makes sense;
> PMTs differ quite a lot (no numbers here, unfortunately), but I am sure
> also Hamamatsu selects them before sending them to vendors. A commercial
> response would be interesting here!
>
> 4) Good question, I am also interested in that one. As already mentioned,
> voltage offers a wider range, but at a certain level S/N gets down, here
> one could think about the gain instead. But every time I used it in the
> past (Zeiss "Amplifier Gain", Olympus "Gain") I was quite disappointed...
> and Leica doesn't have "Gain" at all.
>
> 5) Olympus and Zeiss have it for PMTs, and it's also available in FCS
> upgrades with APDs. Older PMT systems where maybe to noisy for this?!
>
> Michael
>
>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Dear all,
>>
>> I have encountered a good deal of folklore regarding PMT operation in
>> scanning microscopy. Maybe someone in the list can confirm or debunk
>> these informations.
>>
>> 1) Some insist that PMTs should never be turned on and off during an
>> experiment, but instead that their voltage should be turned way down
>> when not in use. Is that true?
>>
>> 2) We have routinely exposed accidentally our PMTs to ambient light
>> (sometime for a few minutes at time). The dark noise should have
>> increased. Is this irreversible? Does sensitivity take a hit as well?
>> We use Hamamatsu PMTs with multialkali (R6357) or GaAs  photocathodes
>> (H7422-40).
>>
>> 3) Some microscope vendors claim that they hand-pick their PMTs from a
>> large lot. Does this make a difference?
>>
>> 4) What is the advantage of changing the PMT voltage except for
>> providing a crude gain control? Would it be better to always set the
>> voltage to an optimal value for best signal to noise ratio and amplify
>> the output current?
>>
>> 5) Why is photon counting not widely available on commercial scopes,
>> since computers would deal easily with digitized counts from the PMT?
>>
>> Thanks to all.
>>
>>
>> Quoc Thang NGUYEN, Ph.D.
>> Assistant Project Scientist
>> Physics Department, UCSD
>> e-mail: [hidden email]
>> Cell Phone: [phone]
>> Fax: [phone]