http://confocal-microscopy-list.275.s1.nabble.com/quantitative-confocal-microscopy-tp5349185p5352286.html
There are 2 different types of quantification. Relative or Absolute. Relative quantification can be made in a regular imaging facility. Ion (ratio-metric) imaging brings some coarse absolute quantification from a relative/comparative ones and can therefore be made almost everywhere, assuming that you know what you are doing. The problem is that most of the times this last request in not achieved.
Regarding absolute quantification you will need to go for single/few molecule imaging, fcs, etc and then, you are right, it is not so common to have expertise on this field as a service. We are running a project to get coarse absolute quantification from a relative one using math and a calibrated light source but still not sure if we will have the time/expertise for doing this. For now it uses loci to get spectral info and works for SP5 and 510 but only relative (benchmarking).
Tel . +351 4464538
Fax. +351 4407970
> Dear Confocalists,
>
> I have a feeling that Confocalists have a strong wish to picture confocal imaging in very bright tones.
>
> Accurate quantitative imaging on a standard (not custom built) confocal scope is a very tough job. And I have very serious doubts that biologists on their own can handle it professionally.
>
> Sandrine, I suggest you to contact a large BioImaging Facility where mathematicians, physicists with some basic knowledge of biology work together with biologists, who are also part of the staff at the BioImaging Facility. Unfortunately, it might not be an easy task to find such a Facility.
>
> MPI in Dresden? Janelia Farm???? any more?
>
> Good luck,
>
> Vitaly
>
>
> From: George McNamara <
[hidden email]>
> To:
[hidden email]
> Sent: Wed, July 28, 2010 9:55:54 PM
> Subject: Re: quantitative confocal microscopy
>
> Hi Sandrine,
>
> Detector gain (PMT voltage) - ideally would like to be a modest value, such as 600 or 700.
>
> Amplifier offset - adjust so that no laser light results in all values being above zero. Note that if you change scan speed and/or detector gain, the offset value(s) may change. When using 12-bit data mode, I typically aim for an offset of ~100, but am happy with anything less than 200 (at moderate gain).
>
> Amplifier gain - you are correct. Do not change from 1.0. It is just does post acquisition arithmetic.
>
> Other items:
>
> You did not mention what fluorophore(s) or fluorescent protein you are using. Or whether you are using the best filter or other wavelength setting(s) for these. Depending on the cell's autofluorescence, this may need optimization.
>
> You did not mention laser power:
>
> * Make sure the laser output is stable! Both within a frame and over experiment time. A starting point is to turn on the transmitted light detector (which as a bonus gets you the cell morphology) and see that you are getting stable intensity. I recommend no polarizing elements in the light path. If you keep your confocal system off at night, may take many hours for the laser output to stabilize (I have never been sure whether it is the lasers or the AOTF intensity controller as being the guilty party or parties).
>
> * If you are using an argon visible laser on an LSM510, run the laser at 6.1 Amps and leave the laser power dialog open (it might fluctuate on its own between 5.9 and 6.3 Amps - that is ok). On our system I've seen the power change to crazy values, like 7.9 Amps, apparently on its own (also on hitting re-use, even though I know the saved setting was not that value). On the LSM710 Argon laser's remote, turn the knob so it is in the middle of the green light range.
>
> * Laser power to the specimen ... lower is better! For example, cardiomyocytes die a spectacular death after a few seconds of 488 nm excitation when loaded with Fluo-4 and imaged at high power.
>
> If you are using an -AM conjugate, more is not better. Besides the possibility of turning your Calcium ion reporter into a chelator, the -AM leaving group is formaldehyde. Could be worse: GFP maturation produces one molecule of H2O2 (so two for CaMeleon), and red fluorescent protein maturation may produce two H2O2's.
>
>
> No pixel in the background at zero ... correct, this is ideal. However, at high gain (say 900 volts) and an offset such that no laser light results in average intensity value around 100 (12-bit mode), when you are imaging with the laser and specimen, if you acquire enough images, eventually statistics dictate that some outlier will reach zero. So what! When you use the "no light" image (or an average of a whole bunch of them) in background subtraction, lots of values outside the cells will hit zero.
>
> A more important item is getting the detector gain and laser power optimized so you can use most of the dynamic range of the confocal to get the data you need. This requires guesswork or using the first specimen to get maximum intensity (typically, saturated Calcium ion with ionophore).
>
> Best wishes,
>
> George
>
>
> At 09:05 AM 7/28/2010, you 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
>
>
>
>
>
>
>
> George McNamara, Ph.D.
> Image Core Manager
> Analytical Imaging Core Facility
> University of Miami, Miller School of Medicine
> Miami, FL 33136
>
[hidden email]
>
[hidden email]
> 305-243-8436 office
>
http://www.sylvester.org/AICF (Analytical Imaging Core Facility)
>
http://www.sylvester.org/AICF/pubspectra.zip (the entire 2000+ spectra .xlsx file is in the zip file)
>
http://home.earthlink.net/~geomcnamara>
>