http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185p5349867.html
Sorry Carl, no VISA. Don't feed the banks, it only encourages them.
On Jul 28, 2010, at 9:51 AM, Vergara, Leoncio A. wrote:
> I just sent you a reply to the e-mail listed. I am interested
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Glen MacDonald
> Sent: Wednesday, July 28, 2010 11:43 AM
> To:
[hidden email]
> 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
> (206) 616-4156
>
[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/030>> 6484684
>>
>> 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