Re: FV1000 : HV vs gain

[Florian Eich]

Dear Listers,
to clarify the HV vs. Gain on FluoView FV1000 topic and to follow Stanislav
Vithas request for someone from Olympus to answer:

1. Adjust HV of the PMT. This regulates how many counts are generated by a
photon hitting the PMT. When you have a weak sample with a good S/N you
can raise the HV to a value where the brightest Point on your image generates
4095 counts, therefore using the complete dynamic range. (Of course also
with a strong sample it is desirable to use the complete dynamic range for your
sample, it simple generates a better data-quality).
But be aware: If you raise the HV too much you reach the non-linear range of
the PMT, leading to an unequal amplification of areas of different brightness.

2. The Gain is an alternative way to facilitate the full dynamic range of the AD-
Converter. To give an example: By using only the HV your sample has intensity
values from 0-1000 counts, rising the HV to top end would take you into the
nonlinear range of the PMT. If this signal is converted by the ADC, you do not
use the whole dynamic range of 4096. If you add a Gain of e.g. 4, you get
everything multiplied by 4 – the PMT noise as well as the actual analog signal.
The PMT noise will be higher, but now you can use a much larger dynamic
range – up to 4095 without sacrificing the linearity of the image. Another
advantage is that the quantization noise will be smaller in comparison to the
signal.

Plainly, if you have a weak sample that can not be imaged with the full
dynamic range in the  linear range using the HV, you can use Gain to amplify
the image and to keep the HV in the linear range. If you don´t care about
using the full dynamic range, there is no need to use the Gain.

Best regards
Florian Eich
Olympus Life Science Europa GmbH

[Julian Smith III]

Thanks for the very clear explanation of the difference!
How does one determine where one's PMT's become non-linear?
Julian

Florian Eich wrote:

> Dear Listers,
> to clarify the HV vs. Gain on FluoView FV1000 topic and to follow Stanislav
> Vithas request for someone from Olympus to answer:
>
> 1. Adjust HV of the PMT. This regulates how many counts are generated by a
> photon hitting the PMT. When you have a weak sample with a good S/N you
> can raise the HV to a value where the brightest Point on your image generates
> 4095 counts, therefore using the complete dynamic range. (Of course also
> with a strong sample it is desirable to use the complete dynamic range for your
> sample, it simple generates a better data-quality).
> But be aware: If you raise the HV too much you reach the non-linear range of
> the PMT, leading to an unequal amplification of areas of different brightness.
>
> 2. The Gain is an alternative way to facilitate the full dynamic range of the AD-
> Converter. To give an example: By using only the HV your sample has intensity
> values from 0-1000 counts, rising the HV to top end would take you into the
> nonlinear range of the PMT. If this signal is converted by the ADC, you do not
> use the whole dynamic range of 4096. If you add a Gain of e.g. 4, you get
> everything multiplied by 4 – the PMT noise as well as the actual analog signal.
> The PMT noise will be higher, but now you can use a much larger dynamic
> range – up to 4095 without sacrificing the linearity of the image. Another
> advantage is that the quantization noise will be smaller in comparison to the
> signal.
>
> Plainly, if you have a weak sample that can not be imaged with the full
> dynamic range in the  linear range using the HV, you can use Gain to amplify
> the image and to keep the HV in the linear range. If you don´t care about
> using the full dynamic range, there is no need to use the Gain.
>
> Best regards
> Florian Eich
> Olympus Life Science Europa GmbH
>
>  

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

803-323-2111 x6427 (vox)
803-323-3448 (fax)
803-524-2347 (cell)

[Barbara Foster]

Hi, Julian

One exercise we used to do when I was supporting the short-lived microspectrometry program at Cambridge/Reichert Jung was to use a 4 quadrant calibration system. It was actually a fluorescent plate with neutral density material of 3 different, known optical densities coated on it.  We would take a reading of the clear + 3 nd filter areas then plot it.  It was pretty evident where the PMT became non-linear.  That information was automatically fed into the system and became part of the system correction algorithm.

Hope this is helpful.

Barbara Foster, President
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]  Skype: fostermme
W: www.MicroscopyEducation.com

NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And don't forget:  MME is now scheduling customized, on-site courses through March 2009.  Call me for a free assessment and quote.

At 07:27 AM 10/8/2008, you wrote:

Thanks for the very clear explanation of the difference!
How does one determine where one's PMT's become non-linear?
Julian

Florian Eich wrote:

Dear Listers,
to clarify the HV vs. Gain on FluoView FV1000 topic and to follow Stanislav Vithas request for someone from Olympus to answer:

1. Adjust HV of the PMT. This regulates how many counts are generated by a photon hitting the PMT. When you have a weak sample with a good S/N you can raise the HV to a value where the brightest Point on your image generates 4095 counts, therefore using the complete dynamic range. (Of course also with a strong sample it is desirable to use the complete dynamic range for your sample, it simple generates a better data-quality).
But be aware: If you raise the HV too much you reach the non-linear range of the PMT, leading to an unequal amplification of areas of different brightness.
2. The Gain is an alternative way to facilitate the full dynamic range of the AD-
Converter. To give an example: By using only the HV your sample has intensity values from 0-1000 counts, rising the HV to top end would take you into the nonlinear range of the PMT. If this signal is converted by the ADC, you do not use the whole dynamic range of 4096. If you add a Gain of e.g. 4, you get everything multiplied by 4 ­ the PMT noise as well as the actual analog signal. The PMT noise will be higher, but now you can use a much larger dynamic range ­ up to 4095 without sacrificing the linearity of the image. Another advantage is that the quantization noise will be smaller in comparison to the signal.
Plainly, if you have a weak sample that can not be imaged with the full dynamic range in the  linear range using the HV, you can use Gain to amplify the image and to keep the HV in the linear range. If you don´t care about using the full dynamic range, there is no need to use the Gain.

Best regards
Florian Eich
Olympus Life Science Europa GmbH

 

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

803-323-2111 x6427 (vox)
803-323-3448 (fax)
803-524-2347 (cell)

[G. Esteban Fernandez]

If you lack samples of known OD you can vary fluorescence intensity by varying the intensity of the excitation laser light, then plot fluorescence vs. laser % to check linearity.  This assumes that the AOTF that regulates laser power is linear but this is a reasonable assumption, see "Quantitative Fluorescence", Ch. 14 in Optical Imaging Techniques in Cell Biology, by Guy Cox (2007).

 

-Esteban

 

On Wed, Oct 8, 2008 at 12:12 PM, Barbara Foster <[hidden email]> wrote:

Hi, Julian

One exercise we used to do when I was supporting the short-lived microspectrometry program at Cambridge/Reichert Jung was to use a 4 quadrant calibration system. It was actually a fluorescent plate with neutral density material of 3 different, known optical densities coated on it.  We would take a reading of the clear + 3 nd filter areas then plot it.  It was pretty evident where the PMT became non-linear.  That information was automatically fed into the system and became part of the system correction algorithm.

Hope this is helpful.

Barbara Foster, President
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]  Skype: fostermme
W: www.MicroscopyEducation.com

NEWS! Visit the NEW and IMPROVED www.MicroscopyEducation.com! And don't forget:  MME is now scheduling customized, on-site courses through March 2009.  Call me for a free assessment and quote.

At 07:27 AM 10/8/2008, you wrote:

Thanks for the very clear explanation of the difference!
How does one determine where one's PMT's become non-linear?
Julian

Florian Eich wrote:

Dear Listers,
to clarify the HV vs. Gain on FluoView FV1000 topic and to follow Stanislav Vithas request for someone from Olympus to answer:

1. Adjust HV of the PMT. This regulates how many counts are generated by a photon hitting the PMT. When you have a weak sample with a good S/N you can raise the HV to a value where the brightest Point on your image generates 4095 counts, therefore using the complete dynamic range. (Of course also with a strong sample it is desirable to use the complete dynamic range for your sample, it simple generates a better data-quality).
But be aware: If you raise the HV too much you reach the non-linear range of the PMT, leading to an unequal amplification of areas of different brightness.
2. The Gain is an alternative way to facilitate the full dynamic range of the AD-
Converter. To give an example: By using only the HV your sample has intensity values from 0-1000 counts, rising the HV to top end would take you into the nonlinear range of the PMT. If this signal is converted by the ADC, you do not use the whole dynamic range of 4096. If you add a Gain of e.g. 4, you get everything multiplied by 4 ­ the PMT noise as well as the actual analog signal. The PMT noise will be higher, but now you can use a much larger dynamic range ­ up to 4095 without sacrificing the linearity of the image. Another advantage is that the quantization noise will be smaller in comparison to the signal.
Plainly, if you have a weak sample that can not be imaged with the full dynamic range in the  linear range using the HV, you can use Gain to amplify the image and to keep the HV in the linear range. If you don´t care about using the full dynamic range, there is no need to use the Gain.

Best regards
Florian Eich
Olympus Life Science Europa GmbH

 

--
Julian P.S. Smith III
Director, Winthrop Microscopy Facility
Dept. of Biology
Winthrop University
520 Cherry Rd.
Rock Hill, SC  29733

803-323-2111 x6427 (vox)
803-323-3448 (fax)
803-524-2347 (cell)

--
G. Esteban Fernandez, Ph.D.
Associate Director
Molecular Cytology Core Facility
University of Missouri
120 Bond Life Sciences Center
Columbia, MO  65211

http://www.biotech.missouri.edu/mcc/

573-882-4895
573-884-9395 fax

[Mark Cannell]

I think this question has been addressed incorrectly IMHO.

Changing the digital gain does not do anything to the recorded signal
except scale it up -noise and all. (IMHO, this a useless feature and I
have never ever used it on _any _ confocal I've used). Now the pmt is
intrinsically a *very* linear device. This was well established in the
1960's by extensive tests by EMI. The linearity of pmts was essential
for the precise stellar measurements being made by astronomers.
Non-linear behavior may be introduced by poor application design, in
particular dynode chain design leaving to dynode voltage droop or local
overload of electrodes (cathode, dynodes &/or anode) and exceeding
manufacturer limits (which will burn the coatings). In fact, the pmt
itself is generally more linear than the current-to-voltage amplifier
and A/D converter -provided  you do not exceed design limits.

A few numbers may help make this clear. If you want < 0.1% non-linearity
total tube current cannot exceed 0.1% of dynode chain current which is
typically 1mA (it may be somewhat less than this as gain is only linear
on a log log plot only and is different for each tube). This implies a
maximum acceptable tube current of ~1uA for our application design
point. Assuming a maximum design tube gain of 10^7 (actual tubes can go
higher than this), this gives a detected photon flux = 0.1pA which is  
about 6 x 10^5 photoelectrons per second. As you reduce operating
voltage, the tube gain goes down and the tube current falls in parallel.
So if the manufacturers have correctly set the A/D converter system gain
up so that the detector is linear to 0.1% at maximum gain then this will
NOT change with pmt voltage (unless you can exceed the maximum design
voltage/gain for the system). This also means that regardless of the
scene brightness, the maximum non-linearity is 0.1% provided no pixels
are saturated. At a dwell time of 1us per pixel this maximum gain  
implies only 0.6 counts per pixel (/us) for this design example (I
suggest there is NO point scaling this value to 4096...).

The bottom line is that detector linearity will not change with pmt
votage if you always have the same A/D converter gain. Post
multiplication of the A/D value does nothing to the scene information.
In dim images where high gain is used, 8 bit recording is perfectly
adequate ('cos less than 100 photoelectrons per pixel) and gives smaller
file sizes. If the scene has a dynamic range of 4000 peak average photon
count per pixel would then have to be >1000 (from almost no saturated
pixels). This represents a huge photon flux at a 1us dwell time but
might be achieved with slower scans. (It is less clear whether it would
be better to scan fast and average).

If there is serious non-linearity in the detector of the Olympus, it can
only be due to bad electrical design (and it needs to be fixed by
redesign) and changing pmt voltage will not alter it. If the light is
too concentrated on the photocathode so that local saturation occurs
then that would need fixing too.  I suspect that  neither of these
problems are actually present  and the original poster is in error. All
3 confocal systems (non-Olympus) I have seen inside have had non-zener
controlled k-d1 dynode steps implying they are being used far from
currents where dynode droop (and linearity) becomes a problem.

My advice is to ignore the digital gain and set pmt voltage to give the
desired gain -and remember to set the black level correctly or your
image really will be non-linear.
 
My 20c

Regards Mark Cannell


  Julian Smith III wrote:

> Thanks for the very clear explanation of the difference!
> How does one determine where one's PMT's become non-linear?
> Julian
>
> Florian Eich wrote:
>> Dear Listers,
>> to clarify the HV vs. Gain on FluoView FV1000 topic and to follow
>> Stanislav Vithas request for someone from Olympus to answer:
>>
>> 1. Adjust HV of the PMT. This regulates how many counts are generated
>> by a photon hitting the PMT. When you have a weak sample with a good
>> S/N you can raise the HV to a value where the brightest Point on your
>> image generates 4095 counts, therefore using the complete dynamic
>> range. (Of course also with a strong sample it is desirable to use
>> the complete dynamic range for your sample, it simple generates a
>> better data-quality).
>> But be aware: If you raise the HV too much you reach the non-linear
>> range of the PMT, leading to an unequal amplification of areas of
>> different brightness.
>> 2. The Gain is an alternative way to facilitate the full dynamic
>> range of the AD-
>> Converter. To give an example: By using only the HV your sample has
>> intensity values from 0-1000 counts, rising the HV to top end would
>> take you into the nonlinear range of the PMT. If this signal is
>> converted by the ADC, you do not use the whole dynamic range of 4096.
>> If you add a Gain of e.g. 4, you get everything multiplied by 4 – the
>> PMT noise as well as the actual analog signal. The PMT noise will be
>> higher, but now you can use a much larger dynamic range – up to 4095
>> without sacrificing the linearity of the image. Another advantage is
>> that the quantization noise will be smaller in comparison to the signal.
>> Plainly, if you have a weak sample that can not be imaged with the
>> full dynamic range in the  linear range using the HV, you can use
>> Gain to amplify the image and to keep the HV in the linear range. If
>> you don´t care about using the full dynamic range, there is no need
>> to use the Gain.
>>
>> Best regards
>> Florian Eich
>> Olympus Life Science Europa GmbH
>>
>>  
>
>

[Florian Eich]

Dear Listers,

maybe my latest post was not clear enough, or even misleading, I am very
sorry for the inconvenience.
To answer on the comments on HV vs gain topic and the following posts about
PMT linearity:
In my previous post I wrote “If you raise HV too much you reach the non-
linear range of the PMT.” I guess that my improper and imprecise use of the
term linearity in this context caused confusion. Saturation would have been
the right term. Please accept my excuses.

The PMTs in FluoView FV1000 work in their linear range.
A linear relation of gain vs supply voltage on the PMT is given from 300 to
1250V.
I tried to attach a graph, but list-rules prevent this. Feel free to contact me
to get the graph.

Please do not mistake this for the gain which you can modulate in the
software.
The gain you modulate in the software is an amplification of the signal AFTER
the PMT but BEFORE the AD-Converter, so, like Marc Cannell rightly stated, is
a pure amplification by a set factor. But you do not multiply the quantisation
error of the ADC because the amplification takes place before AD conversion.

I hope this answered any open questions.

Best Regards
Florian Eich
Olympus Life Science Europa GmbH