How do gain and offset affect a confocals hardware?

[Matthew Pearson-3]

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Hi,

I am constantly playing around with the gain and offset settings on our confocal but I feel I should actually know what is actually happening to the hardware in order to raise the image gain.  I know that the photocathode in the PMT coverts photons to electrons which are multiplied as the electrons contact the dynodes, so does raising the gain increase the number of electrons and if so how does this happen? But I have no idea how the offset is generated.

Best wishes,

Matt Pearson.

[Michael Weber-4]

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Matt,

do you mean detector voltage or gain? Which system are we talking about?

Regarding the photomultiplier and the influence of detector voltage, this
might be of interest:
http://en.wikipedia.org/wiki/Photomultiplier

The offset, to my understanding, is just a threshold that defines which is
the lowest intensity that will be displayed. So ideally the lowest signal
should be placed on the first intensity level (1).

Michael

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>  Hi,
>
>  I am constantly playing around with the gain and offset settings on our
> confocal but I feel I should actually know what is actually happening to
> the hardware in order to raise the image gain.  I know that the
> photocathode in the PMT coverts photons to electrons which are multiplied
> as the electrons contact the dynodes, so does raising the gain increase
> the number of electrons and if so how does this happen? But I have no
> idea how the offset is generated.
>
>  Best wishes,
>
>  Matt Pearson.
>
>
>     --  Imaging Technician Cell Biology Division Institute of
> Ophthalmology University College London EC1V 9EL 020 7608 6857
> [hidden email]

[mmodel]

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Dear Matt:

 

My understanding is that PMT generates voltage and passes it on to an electronic circuit. Additionally, you may subtract an offset voltage from the PMT output. Then an electronic amplifier can multiply all that by a constant factor.

The general relationship between the PMT readout and parameters is

Signal = (A-B*Offset)*Gain + Cv*Gain*E

Here A and B are coefficients, C_V is the voltage-dependent amplification of PMT (depends on voltage non-linearly, as pV^k) and E is the intensity coming from the sample. As long as Offset ≤ A/B, the first addend in this equation is equal to the dark level. However, if the offset is set too high (greater than A/B), the dark level does not enter the equation for the signal in a simple way and non-linearity may occur, especially at high gain and low intensity levels.

 

Mike Model

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Matthew Pearson
Sent: Friday, March 14, 2008 10:28 AM
To: [hidden email]
Subject: How do gain and offset affect a confocals hardware?

 

Hi,

I am constantly playing around with the gain and offset settings on our confocal but I feel I should actually know what is actually happening to the hardware in order to raise the image gain.  I know that the photocathode in the PMT coverts photons to electrons which are multiplied as the electrons contact the dynodes, so does raising the gain increase the number of electrons and if so how does this happen? But I have no idea how the offset is generated.

Best wishes,

Matt Pearson.

-- 

Imaging Technician

Cell Biology Division

Institute of Ophthalmology

University College London

EC1V 9EL

020 7608 6857

[hidden email]