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Hey Folks --
One more note on linearity: We've found that on our Leica SP1 (8-bit system) that the response of the PMTs is only linear between 20 and 200 grey levels (these limits depend on how fussy one is about curve-fitting). Same results whether we use a graded concentration of dye in solution, or adjust excitation with the AOTF (assuming AOTF attenuation is linear). Does anyone know if this is typical?
Peace! Greg.
Greg Martin
Keck Imaging Center
University of Washington
206-685-8784
www.depts.washington.edu/keck
----- Original Message -----
From: "Robert Atkinson" <[log in to unmask]>
To: <[log in to unmask]>
Sent: Wednesday, November 10, 2004 2:06 AM
Subject: Re: PMT sensitivity
> Hi Carl,
> Mark and Guy have already answered this and I'd agree with what they
> say. Basically once the system is stabilised at a reasonable gain
> setting the PMT will be linear to much less than 1 count of your 8 bit A
> to D. Assuming the amplifiers are reasonable you should have no problem.
> On the subject of variation between tubes, we have our PMT's specially
> selected by the manufacturer for better matching.
> One other factor that can cause problems is if the PMT has been exposed
> to excessive light levels. This causes problems even if the tube is not
> powered at the time. A PMT can take many hours to recover from even a
> few seconds of exposure to room lighting.
>
> Regards,
> Robert.
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On
> Behalf Of carl
> Sent: 09 November 2004 17:26
> To: [log in to unmask]
> Subject: Re: PMT sensitivity
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Robert,
> Thanks very much for the reply. However my background doesn't include
> the
> wherewithall to gleen from your reply the answer to my question.
>
> I will rephrase: At a given gain setting, do pixel values from 0-255 in
> an
> image imply a linear increase in brightness in the sample? If not, how
> does
> one correct for the non-linearity? When the gain is changed to a
> different
> setting, is there a change in the slope/shape of the sensitivity curve?
> The
> user that prompted this question wants to measure greyscale values and
> wants
> to know if a range of low values have the same relationship to each
> other as
> mid-values have to each other and high pixel values have to each other.
> I
> guess the next question is, "Is there a simple answer to this question?"
>
> You identified several unknowns regarding how the PMT is being used in
> the
> system. These questions could probably be best be answered by the Nikon
> folks who made the system, however I've received no response yet from a
> direct query. Is anyone out there?
>
> Thanks again,
> Carl
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> University of Arizona
> 520-626-8469
> FAX 520-621-3709
> ----- Original Message -----
> From: "Robert Atkinson" <[log in to unmask]>
> To: <[log in to unmask]>
> Sent: Tuesday, November 09, 2004 3:59 AM
> Subject: Re: PMT sensitivity
>
>
>> Hi Carl,
>> I'm not sure what exactly you are asking. If you mean that if you
> change
>> the anode voltage from 400V to 500V and the gain goes from 2Alm to
>> 10A/lm, will changing it from 500 to 600V give a gain of 50A/lm then
> the
>> answer is no the gain will be 30A/lm, you'd need to go to 700V to get
>> 50A/lm. The graph "Anode Luminous Sensitivity and Gain
> Characteristics"
>> on the data sheet (fig 2 on my copy) shows this. It unfortunately a
> very
>> cluttered graph with non linear axes.
>> The other important question is what "mode" the PMT is being used in,
>> I.E single photon or current mode. If the light level is so low that
> you
>> are getting individual pulses (one for each photon) from the PMT then
>> adjusting the gain will not affect the number of pulses, just their
>> amplitude. If you are in current mode then the gain will directly
> affect
>> the signal level.
>> I do not know how the PCM2000 operates, but may instruments use a
>> current amplifier with a relatively long time constant. This when
>> individual pulses are present it integrates them and changes to
> current
>> mode when the out put becomes continuous. Have you tried imaging a
>> dilution series at different gain settings. Again I don't know the
>> PCM2000 but normally the "gain" control just adjusts the PMT anode
>> voltage. The other unknown is whether the ratio between the gain
>> "control" and the voltage is linear. Some instruments scale the
> voltage
>> change so that 10% change in the control really is 10% change in gain.
> A
>> dilution series check should show this.
>>
>> HTH,
>> Robert.
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On
>> Behalf Of carl
>> Sent: 09 November 2004 00:04
>> To: [log in to unmask]
>> Subject: PMT sensitivity
>>
>> Search the CONFOCAL archive at
>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>> Hi All,
>> Does anyone have info on the linearity of the sensitivity of the
>> Hamamatsu
>> R928 PMT that we have in our Nikon PCM2000 confocal? Does it change
>> with
>> gain settings? I have a 4-page spec sheet on this thing but I can't
>> decipher what it is telling me.
>> Thanks very much,
>> Carl
>>
>> Carl A. Boswell, Ph.D.
>> Molecular and Cellular Biology
>> University of Arizona
>> 520-626-8469
>> FAX 520-621-3709
>>
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