Re: quantitative confocal microscopy

Posted by Boswell, Carl A - (cboswell) on
URL: http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185p5349817.html

Do you take Visa?
C

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Glen MacDonald" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, July 28, 2010 9:42 AM
Subject: Re: quantitative confocal microscopy


This may be of interest to the community and I apologize anyone is offended
by the slightly commercial nature.

Alan Hibbs had shipped copies of his book to me in anticipation of a
teaching engagement in North America.  Unfortunately, he unexpectedly passed
away before he could visit.  I've still got a dozen copies for US$125,
shipping by US mail is about $12.  Proceeds are sent to his family.  If
interested, please reply directly to my home email, [hidden email].


Regards,
Glen

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]








On Jul 28, 2010, at 6:25 AM, John Oreopoulos wrote:

> Hi Sandrine, your question is sure to draw answers as it has been asked
> several times before. Quantitative fluorescence microscopy is not trivial,
> but can be somewhat reliable if you try to carefully control as many
> experimental parameters as possible. Other people on this list server can
> give you more details, but as a quick reference, try reading Jim Pawley's
> "39 steps":
> http://labs.pbrc.edu/cellbiology/documents/39steps.pdf
>
> As for detector gain and offset settings, Alan Hibbs' book has a nice
> section that outlines a good method for determining the best settings:
> http://www.amazon.com/Confocal-Microscopy-Biologists-Alan-Hibbs/dp/0306484684
>
> All the best,
>
> John Oreopoulos
>
>
>
> On 2010-07-28, at 9:05 AM, Sandrine Pouvreau wrote:
>
>> Hello.
>> I had this discussion with several colleagues (biologist like me), and
>> did some
>> research on my own, but I figurate the best would be to submit the
>> question
>> to this list. Here’s the point: we are doing quantitative measurement
>> with
>> confocal microscopy (calcium measurement) using a Zeiss LSM exciter.
>> There
>> are 3 parameters of the PMT that can be configured: detector gain,
>> amplifier
>> offset, amplifier gain. The only parameter I adjust to improve the signal
>> is the
>> detector gain. I keep the amplifier gain at 1 as I read in several papers
>> that
>> increasing it will not improve the signal over noise ratio (they also say
>> that it
>> is bad for several reasons that I can not summarize here). Is that
>> correct? I
>> put the offset usually at zero. I saw that a change in offset can affect
>> the
>> calcium signal. In any case, I keep the same offset in a series of
>> records that
>> I whish to compare. Any comment on this? My guess is that the same should
>> be done with the amplifier gain.
>> Ah, a last point: the offset should be tuned so no pixel in the
>> background has
>> the value of zero right?
>> Does anybody have any clarification for these questions? Thanks for your
>> help.
>>
>> Regards
>> Sandrine