LM Hybrid vs PMT non-descan detectors (NDDs) for multi-photon microscopy

[Smith, Benjamin E.]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hey listers,

I was wondering if anyone has used hydrid detectors in the NDD position
on a multi-photon microscope, and if so, what they thought of them.

Right now we have PMT NDDs installed on our Leica SP8, but we are
considering upgrading to HyD detectors.  We have a couple of HyDs in the
descan position, and they are far more sensitive than PMTs, but they
also can shut-off when the fluorescent signal is too bright. Normally
this is not an issue, but for large tiling runs it can be prohibitively
time consuming to check the gain on the detector for the entire imaging
volume, and yet it is equally frustrating to have a detector quit 3/4 of
the way into an hour long tiling run due to an unexpectedly bright cell,
or worse yet to have a detector quit during live imaging because a cell
became increasingly bright.

We would love the increased sensitivity of HyD NDDs, especially for live
imaging and autofluorescence detection, but I'm not sure if the tendency
of the HyD detectors to shut-off makes HyD NDDs more of a hassle then
they're worth.

Thanks for any feedback,
   Ben Smith

--
Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory
Research Scientist II
University of Oklahoma
Norman, OK 73019
E-mail: [hidden email]
Voice   405-325-4391
FAX  [phone]
http://www.microscopy.ou.edu/

[Steffen Dietzel]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Ben,

sorry I can't give you any advice, but you did make an excellent
argument why companies should implement "Controled light exposure
microscopy" as suggested by Erik Manders. If the Laser would
automatically be attenuated when the image gets very bright, this could
solve the problem.

Question: Would the Hybrid detector stay off for the rest of the scan?
On our LaVisionBiotech multi-Photon, the GaAsps also shut automatically
off, but are turned on again after a moment. So you don't loose the rest
of the stack/time series but end up only with a few black image lines
instead.

Steffen

On 24.06.2013 21:12, Ben Smith wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hey listers,
>
> I was wondering if anyone has used hydrid detectors in the NDD position
> on a multi-photon microscope, and if so, what they thought of them.
>
> Right now we have PMT NDDs installed on our Leica SP8, but we are
> considering upgrading to HyD detectors. We have a couple of HyDs in the
> descan position, and they are far more sensitive than PMTs, but they
> also can shut-off when the fluorescent signal is too bright. Normally
> this is not an issue, but for large tiling runs it can be prohibitively
> time consuming to check the gain on the detector for the entire imaging
> volume, and yet it is equally frustrating to have a detector quit 3/4 of
> the way into an hour long tiling run due to an unexpectedly bright cell,
> or worse yet to have a detector quit during live imaging because a cell
> became increasingly bright.
>
> We would love the increased sensitivity of HyD NDDs, especially for live
> imaging and autofluorescence detection, but I'm not sure if the tendency
> of the HyD detectors to shut-off makes HyD NDDs more of a hassle then
> they're worth.
>
> Thanks for any feedback,
> Ben Smith
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[Smith, Benjamin E.]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hey Steffen,
     Unfortunately, our hybrid detectors shut-off for the rest of the
series if a signal is too bright.  However, a representative from Leica
informed me that there are several ways around this including line/frame
acqusition (which would be fine for live imaging, but time prohibitive
for tiling) and using the detectors in photon-counting mode with 12 or
16-bit intensity resolution (which also can be a bit more time consuming).

     They also mentioned that hydrid detectors are effectively always in
photon counting mode, so it makes sense why they shut-down.  If the
moment a SPAD refreshes, it is then hit by another photon, it would burn
out pretty quick from all the current.

     What would be really nice would be if the detector could sense the
frequency of photon detection events, and if the frequency got too high
then the pixel would be flagged as saturated, and then the detector
could stay below the bias voltage until the laser moved on to the next
pixel.  This would make hybrid detectors comparable to PMT detectors in
high intensity situations.

-Ben Smith

On 6/25/2013 3:10 AM, Steffen Dietzel wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Ben,
>
> sorry I can't give you any advice, but you did make an excellent
> argument why companies should implement "Controled light exposure
> microscopy" as suggested by Erik Manders. If the Laser would
> automatically be attenuated when the image gets very bright, this
> could solve the problem.
>
> Question: Would the Hybrid detector stay off for the rest of the scan?
> On our LaVisionBiotech multi-Photon, the GaAsps also shut
> automatically off, but are turned on again after a moment. So you
> don't loose the rest of the stack/time series but end up only with a
> few black image lines instead.
>
> Steffen
>
> On 24.06.2013 21:12, Ben Smith wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hey listers,
>>
>> I was wondering if anyone has used hydrid detectors in the NDD position
>> on a multi-photon microscope, and if so, what they thought of them.
>>
>> Right now we have PMT NDDs installed on our Leica SP8, but we are
>> considering upgrading to HyD detectors. We have a couple of HyDs in the
>> descan position, and they are far more sensitive than PMTs, but they
>> also can shut-off when the fluorescent signal is too bright. Normally
>> this is not an issue, but for large tiling runs it can be prohibitively
>> time consuming to check the gain on the detector for the entire imaging
>> volume, and yet it is equally frustrating to have a detector quit 3/4 of
>> the way into an hour long tiling run due to an unexpectedly bright cell,
>> or worse yet to have a detector quit during live imaging because a cell
>> became increasingly bright.
>>
>> We would love the increased sensitivity of HyD NDDs, especially for live
>> imaging and autofluorescence detection, but I'm not sure if the tendency
>> of the HyD detectors to shut-off makes HyD NDDs more of a hassle then
>> they're worth.
>>
>> Thanks for any feedback,
>> Ben Smith
>>
>
>

[Zdenek Svindrych]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi Ben,

you can use the fastest possible scanning (preferably resonant) and average
(or sum) several dim images. But I remember that the FOV shrinks appreciably
when the normal scanner (accidentally being referred to as a "large FOV
scanner") is pushed to the limits...

Your suggested per-pixel HyD voltage is not practical (nor possible perhaps)
as one cannot change the high voltage (6kV) quickly enough (up to about 10
MHz), and very accurately and predictably at the same time. On the other
hand, it is not necessary to turn off the detector for the rest of the
experiment... A few black lines is more like the kind of behavior I'd
expect. Leica definitively should improve upon this.

Good luck with your acquisition!

Zdenek Svindrych




---------- Původní zpráva ----------
Od: Ben Smith <[hidden email]>
Datum: 25. 6. 2013
Předmět: Re: LM Hybrid vs PMT non-descan detectors (NDDs) for multi-photon
microscopy

"*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hey Steffen,Odeslat zprávu(https://email.seznam.cz/#)
Unfortunately, our hybrid detectors shut-off for the rest of the
series if a signal is too bright. However, a representative from Leica
informed me that there are several ways around this including line/frame
acqusition (which would be fine for live imaging, but time prohibitive
for tiling) and using the detectors in photon-counting mode with 12 or
16-bit intensity resolution (which also can be a bit more time consuming).

They also mentioned that hydrid detectors are effectively always in
photon counting mode, so it makes sense why they shut-down. If the
moment a SPAD refreshes, it is then hit by another photon, it would burn
out pretty quick from all the current.

What would be really nice would be if the detector could sense the
frequency of photon detection events, and if the frequency got too high
then the pixel would be flagged as saturated, and then the detector
could stay below the bias voltage until the laser moved on to the next
pixel. This would make hybrid detectors comparable to PMT detectors in
high intensity situations.

-Ben Smith

On 6/25/2013 3:10 AM, Steffen Dietzel wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Ben,
>
> sorry I can't give you any advice, but you did make an excellent
> argument why companies should implement "Controled light exposure
> microscopy" as suggested by Erik Manders. If the Laser would
> automatically be attenuated when the image gets very bright, this
> could solve the problem.
>
> Question: Would the Hybrid detector stay off for the rest of the scan?
> On our LaVisionBiotech multi-Photon, the GaAsps also shut
> automatically off, but are turned on again after a moment. So you
> don't loose the rest of the stack/time series but end up only with a
> few black image lines instead.
>
> Steffen
>
> On 24.06.2013 21:12, Ben Smith wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hey listers,
>>
>> I was wondering if anyone has used hydrid detectors in the NDD position
>> on a multi-photon microscope, and if so, what they thought of them.
>>
>> Right now we have PMT NDDs installed on our Leica SP8, but we are
>> considering upgrading to HyD detectors. We have a couple of HyDs in the
>> descan position, and they are far more sensitive than PMTs, but they
>> also can shut-off when the fluorescent signal is too bright. Normally
>> this is not an issue, but for large tiling runs it can be prohibitively
>> time consuming to check the gain on the detector for the entire imaging
>> volume, and yet it is equally frustrating to have a detector quit 3/4 of
>> the way into an hour long tiling run due to an unexpectedly bright cell,
>> or worse yet to have a detector quit during live imaging because a cell
>> became increasingly bright.
>>
>> We would love the increased sensitivity of HyD NDDs, especially for live
>> imaging and autofluorescence detection, but I'm not sure if the tendency
>> of the HyD detectors to shut-off makes HyD NDDs more of a hassle then
>> they're worth.
>>
>> Thanks for any feedback,
>> Ben Smith
>>
>
>"