Quantifying fluorescence help

[Maria Mazzillo]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my thesis work.  I am
looking for a reliable method for quantifying fluorescence.  

My project deals with marine dinoflagellates (zooxanthellae) that reside intracellularly in
hosts (usually cnidarians).  I am working with cultures or isolates of only the dinoflagellates
for my confocal work.  I have an antibody that was created against the surface secretions
of mucilage (secreted as part of a daily cycle by the alga) from one strain of zooxanthellae.  
I am attempting to use this antibody to label various strains to identify differences in
mucilage between them.  Thus far, I have seen that the strain the antibody was created
against labels around the cell fairly brightly.  Most samples either show this or a complete
lack of labeling.  However, a few samples show a faint fluorescence lifted off the cell
surface.  I would like to be able to quantify this fluorescence in comparison to either the
control strain that the antibody was created against or against a known fluorescence.  

Any help on ideas for this, or places to look for ideas would be greatly appreciated.

Thanks!

Maria Mazzillo

[Rietdorf, Jens]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Maria,

(no commercial interest) Invitrogen sells a product called InSpeck, ie
beads with a calibrated intensity of labelling. They come in different
intensities, like 100%, 50% 25% usf. If you include those in all your
preparations you can normalise your signal to the beads, which may be
sufficient to have a rough idea of the relative brightness of your
labelling.

Cheers, jens

 
-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Maria Mazzillo
Sent: Freitag, 18. Juli 2008 15:26
To: [hidden email]
Subject: Quantifying fluorescence help

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my
thesis work.  I am looking for a reliable method for quantifying
fluorescence.  

My project deals with marine dinoflagellates (zooxanthellae) that reside
intracellularly in hosts (usually cnidarians).  I am working with
cultures or isolates of only the dinoflagellates for my confocal work.
I have an antibody that was created against the surface secretions of
mucilage (secreted as part of a daily cycle by the alga) from one strain
of zooxanthellae.  
I am attempting to use this antibody to label various strains to
identify differences in mucilage between them.  Thus far, I have seen
that the strain the antibody was created against labels around the cell
fairly brightly.  Most samples either show this or a complete lack of
labeling.  However, a few samples show a faint fluorescence lifted off
the cell surface.  I would like to be able to quantify this fluorescence
in comparison to either the control strain that the antibody was created
against or against a known fluorescence.  

Any help on ideas for this, or places to look for ideas would be greatly
appreciated.

Thanks!

Maria Mazzillo

[cromey]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Quantifying fluorescence is difficult to do properly and unfortunately is
quite easy to do poorly.  At my institution I always refer questions of this
sort to Jim Pawley's excellent article (The 39 Steps: A Cautionary Tale
about "quantatative" 3D Fluorescence Microscopy).  If you can control most
of the things Jim mentions, then you are probably more talented/patient than
a lot of us.

See:
http://www.zoology.wisc.edu/faculty/Paw/pdfs/The_39_Steps_corrected.pdf

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Assistant Scientific Investigator
Dept. of Cell Biology & Anatomy, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/exppath/
Home of: "Microscopy and Imaging Resources on the WWW"


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Maria Mazzillo
Sent: Friday, July 18, 2008 6:26 AM
To: [hidden email]
Subject: Quantifying fluorescence help

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my thesis
work.  I am
looking for a reliable method for quantifying fluorescence.  

My project deals with marine dinoflagellates (zooxanthellae) that reside
intracellularly in
hosts (usually cnidarians).  I am working with cultures or isolates of only
the dinoflagellates
for my confocal work.  I have an antibody that was created against the
surface secretions
of mucilage (secreted as part of a daily cycle by the alga) from one strain
of zooxanthellae.  
I am attempting to use this antibody to label various strains to identify
differences in
mucilage between them.  Thus far, I have seen that the strain the antibody
was created
against labels around the cell fairly brightly.  Most samples either show
this or a complete
lack of labeling.  However, a few samples show a faint fluorescence lifted
off the cell
surface.  I would like to be able to quantify this fluorescence in
comparison to either the
control strain that the antibody was created against or against a known
fluorescence.  

Any help on ideas for this, or places to look for ideas would be greatly
appreciated.

Thanks!

Maria Mazzillo

[Julio Vazquez]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

=

Hi Maria, 

It's difficult to give a precise answer without knowing how much you already know or don't know. But basically:

1. Prepare all samples in the same manner; ideally at the same time

2. Get good images (maximize the dynamic range with your microscope system): on a confocal, try to get the background close to, but not equal to zero, and get your highest signals close to the max intensity limit, but not saturated.

3. Collect all images under the same conditions ; ideally, collect them back to back on the same day, so as to minimize any instrument (and operator) variability. Adjust settings for the brightest sample, and use the same for all other samples. If images obtained this way are not good and you need to change imaging parameters between samples, you should either include reference standards (e.g. beads), or do the appropriate calculations to normalize results (but this may be complicated, and not necessarily accurate)

4. Use your favorite software to measure the pixel intensities (total, average, intensity profile, etc, depending on what you need to know) in your regions of interest; ImageJ is great as a start:

http://rsbweb.nih.gov/ij/

If you want to measure labeling intensities in the entire microorganisms, then you should image the whole cells in 3-D,using proper sampling in x, y, and z, and use some 3-D imaging software to get the numbers. You can also do that with ImageJ by analyzing all relevant sections (or projections of all relevant sections)

If you can make it this far, then you should use the additional suggestions (such as Jim Pawley's 39 steps...)

If you can make it that far but still have difficulties, let us know what the specific problem is...

Handling/analyzing digital microscopy images can be difficult, intimidating, and fraught with potential hazards (as you may be aware if you have followed the thread on "image manipulation"). There are many ways to process/analyze images, which are hard to efficiently learn on a forum like this one if you are a complete beginner (we can't give you a class on image analysis). If you have a confocal facility at your institution, make sure you use their expertise in this area.

--

Julio Vazquez

Fred Hutchinson Cancer Research Center

Seattle, WA 98109-1024

http://www.fhcrc.org/

=

On Jul 18, 2008, at 6:25 AM, Maria Mazzillo wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my thesis work.  I am 

looking for a reliable method for quantifying fluorescence.  

My project deals with marine dinoflagellates (zooxanthellae) that reside intracellularly in 

hosts (usually cnidarians).  I am working with cultures or isolates of only the dinoflagellates 

for my confocal work.  I have an antibody that was created against the surface secretions 

of mucilage (secreted as part of a daily cycle by the alga) from one strain of zooxanthellae.  

I am attempting to use this antibody to label various strains to identify differences in 

mucilage between them.  Thus far, I have seen that the strain the antibody was created 

against labels around the cell fairly brightly.  Most samples either show this or a complete 

lack of labeling.  However, a few samples show a faint fluorescence lifted off the cell 

surface.  I would like to be able to quantify this fluorescence in comparison to either the 

control strain that the antibody was created against or against a known fluorescence.  

Any help on ideas for this, or places to look for ideas would be greatly appreciated.

Thanks!

Maria Mazzillo

... [show rest of quote]

[JOEL B. SHEFFIELD]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I would add to this excellent summary that you should become
"quantitatively" aware of fading issues with your fluorphores.  
Although it is possible to reduce the degree of fading with various
mounting media, the rate does not go to zero.  For that reason, you
may have to standardize your observation time so that you capture
images only after x amount of exposure.

I would also stress Julio's point that the images should be captured
BELOW saturation.  Once you saturate an image, all bets are off.

You might also ask yourself what level of precision you need.  Are
you looking for changes of 10%, 50% etc.  

Best of luck,

Joel

>
> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-
> bin/wa?S1=confocal
> =
> Hi Maria,
>
> It's difficult to give a precise answer without knowing how much you
> already know or don't know. But basically:
>
>
> 1. Prepare all samples in the same manner; ideally at the same time
>
> 2. Get good images (maximize the dynamic range with your microscope
> system): on a confocal, try to get the background close to, but not
> equal to zero, and get your highest signals close to the max
> intensity limit, but not saturated.
>
> 3. Collect all images under the same conditions ; ideally, collect
> them back to back on the same day, so as to minimize any instrument
> (and operator) variability. Adjust settings for the brightest sample,
> and use the same for all other samples. If images obtained this way
> are not good and you need to change imaging parameters between
> samples, you should either include reference standards (e.g. beads),
> or do the appropriate calculations to normalize results (but this may
> be complicated, and not necessarily accurate)
>
>
> 4. Use your favorite software to measure the pixel intensities
> (total, average, intensity profile, etc, depending on what you need
> to know) in your regions of interest; ImageJ is great as a start:
>
> http://rsbweb.nih.gov/ij/
>
> If you want to measure labeling intensities in the entire
> microorganisms, then you should image the whole cells in 3-D,using
> proper sampling in x, y, and z, and use some 3-D imaging software to
> get the numbers. You can also do that with ImageJ by analyzing all
> relevant sections (or projections of all relevant sections)
>
>
> If you can make it this far, then you should use the additional
> suggestions (such as Jim Pawley's 39 steps...)
>
> If you can make it that far but still have difficulties, let us know
> what the specific problem is...
>

... [show rest of quote]

--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]  
[phone], fax [phone]
http://astro.temple.edu/~jbs

[Adams,Henry P]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Also, I don't think this was mentioned in the thread. When using a confocal acquire your images at 12-bit for doing 'quantitation', and aren't we really talking about doing semi-quantitation since you are comparing isolates. Quantitation infers you know what the absolute quantities of what you are measuring.

Hank Adams
Microscopy Core
Department of Genetics
U.T. M.D.Anderson Cancer Center
Houston, Tx

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Douglas Cromey
Sent: Friday, July 18, 2008 10:18 AM
To: [hidden email]
Subject: Re: Quantifying fluorescence help

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Quantifying fluorescence is difficult to do properly and unfortunately is
quite easy to do poorly.  At my institution I always refer questions of this
sort to Jim Pawley's excellent article (The 39 Steps: A Cautionary Tale
about "quantatative" 3D Fluorescence Microscopy).  If you can control most
of the things Jim mentions, then you are probably more talented/patient than
a lot of us.

See:
http://www.zoology.wisc.edu/faculty/Paw/pdfs/The_39_Steps_corrected.pdf

Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Assistant Scientific Investigator
Dept. of Cell Biology & Anatomy, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/exppath/
Home of: "Microscopy and Imaging Resources on the WWW"


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Maria Mazzillo
Sent: Friday, July 18, 2008 6:26 AM
To: [hidden email]
Subject: Quantifying fluorescence help

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my thesis
work.  I am
looking for a reliable method for quantifying fluorescence.

My project deals with marine dinoflagellates (zooxanthellae) that reside
intracellularly in
hosts (usually cnidarians).  I am working with cultures or isolates of only
the dinoflagellates
for my confocal work.  I have an antibody that was created against the
surface secretions
of mucilage (secreted as part of a daily cycle by the alga) from one strain
of zooxanthellae.
I am attempting to use this antibody to label various strains to identify
differences in
mucilage between them.  Thus far, I have seen that the strain the antibody
was created
against labels around the cell fairly brightly.  Most samples either show
this or a complete
lack of labeling.  However, a few samples show a faint fluorescence lifted
off the cell
surface.  I would like to be able to quantify this fluorescence in
comparison to either the
control strain that the antibody was created against or against a known
fluorescence.

Any help on ideas for this, or places to look for ideas would be greatly
appreciated.

Thanks!

Maria Mazzillo

[Craig Brideau]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

How good an idea of photo-bleaching would such a bead give you?  I know it will be different from whatever dye is being used in your tissue, but can you still use it somehow to gauge photo-bleaching rates?

Thanks,

Craig

On Fri, Jul 18, 2008 at 8:03 AM, Rietdorf, Jens <[hidden email]> wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Maria,

(no commercial interest) Invitrogen sells a product called InSpeck, ie
beads with a calibrated intensity of labelling. They come in different
intensities, like 100%, 50% 25% usf. If you include those in all your
preparations you can normalise your signal to the beads, which may be
sufficient to have a rough idea of the relative brightness of your
labelling.

Cheers, jens

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Maria Mazzillo
Sent: Freitag, 18. Juli 2008 15:26
To: [hidden email]
Subject: Quantifying fluorescence help

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my
thesis work.  I am looking for a reliable method for quantifying
fluorescence.

My project deals with marine dinoflagellates (zooxanthellae) that reside
intracellularly in hosts (usually cnidarians).  I am working with
cultures or isolates of only the dinoflagellates for my confocal work.
I have an antibody that was created against the surface secretions of
mucilage (secreted as part of a daily cycle by the alga) from one strain
of zooxanthellae.
I am attempting to use this antibody to label various strains to
identify differences in mucilage between them.  Thus far, I have seen
that the strain the antibody was created against labels around the cell
fairly brightly.  Most samples either show this or a complete lack of
labeling.  However, a few samples show a faint fluorescence lifted off
the cell surface.  I would like to be able to quantify this fluorescence
in comparison to either the control strain that the antibody was created
against or against a known fluorescence.

Any help on ideas for this, or places to look for ideas would be greatly
appreciated.

Thanks!

Maria Mazzillo

... [show rest of quote]

[Patrick Van Oostveldt]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hey,

Probably this problem can be tackled with flow cytometry, where  
conditions of measurements can e better controlled.

Patrick Van Oostveldt

Quoting Maria Mazzillo <[hidden email]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I am a PhD student at Auburn University just getting started into my  
>  thesis work.  I am
> looking for a reliable method for quantifying fluorescence.
>
> My project deals with marine dinoflagellates (zooxanthellae) that  
> reside intracellularly in
> hosts (usually cnidarians).  I am working with cultures or isolates  
> of only the dinoflagellates
> for my confocal work.  I have an antibody that was created against  
> the surface secretions
> of mucilage (secreted as part of a daily cycle by the alga) from one  
>  strain of zooxanthellae.
> I am attempting to use this antibody to label various strains to  
> identify differences in
> mucilage between them.  Thus far, I have seen that the strain the  
> antibody was created
> against labels around the cell fairly brightly.  Most samples either  
>  show this or a complete
> lack of labeling.  However, a few samples show a faint fluorescence  
> lifted off the cell
> surface.  I would like to be able to quantify this fluorescence in  
> comparison to either the
> control strain that the antibody was created against or against a  
> known fluorescence.
>
> Any help on ideas for this, or places to look for ideas would be  
> greatly appreciated.
>
> Thanks!
>
> Maria Mazzillo
>

... [show rest of quote]

--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel [phone]
fax [phone]

[Jurriaan Zwier]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Maria,

Quite hard to really quantify your confocal images. We've tried and
devoloped a method using ca 100 nm thin fluorescent layers and used them
for calibrating the total intensities of a z-stack of confocal images. Our
efforts were just published here:
'Quantitative image correction and calibration for confocal fluorescence
microscopy using thin reference layers and SIPchart-based calibration
procedures', J.M. Zwier, L. Oomen, L.Brocks, K.Jalink, G.J. Brakenhoff,
Journal of microscopy 231 (2008) p59-69

kind regards,
Jurriaan

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> I am a PhD student at Auburn University just getting started into my
> thesis work.  I am
> looking for a reliable method for quantifying fluorescence.
>
> My project deals with marine dinoflagellates (zooxanthellae) that reside
> intracellularly in
> hosts (usually cnidarians).  I am working with cultures or isolates of
> only the dinoflagellates
> for my confocal work.  I have an antibody that was created against the
> surface secretions
> of mucilage (secreted as part of a daily cycle by the alga) from one
> strain of zooxanthellae.
> I am attempting to use this antibody to label various strains to identify
> differences in
> mucilage between them.  Thus far, I have seen that the strain the antibody
> was created
> against labels around the cell fairly brightly.  Most samples either show
> this or a complete
> lack of labeling.  However, a few samples show a faint fluorescence lifted
> off the cell
> surface.  I would like to be able to quantify this fluorescence in
> comparison to either the
> control strain that the antibody was created against or against a known
> fluorescence.
>
> Any help on ideas for this, or places to look for ideas would be greatly
> appreciated.
>
> Thanks!
>
> Maria Mazzillo
>

... [show rest of quote]

[Steve Hunter-2]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Maria,

The main issue you will face is photobleaching.  Standardising the
exposure times and the use of calibrated beads will help.   They will
give you relative values where you can at least get a relative but not
absolute result but you will need to be very disciplined and methodical.

Wide field and point scanning confocals have very high bleaching rates.
I would also like to reinforce Julio and Joel's remarks about staying
away from saturation of your fluorophores in excitation. The spinning
disc confocals have vastly lower bleaching rates, at least one order of
magnitude less and because of this your relative determinations should
be better. Look for a system based around the Yokogawa unit say from
Andor of Perkinelmer.  These systems are likely to give you the best
results in my opinion.

As pointed out in other responses there may be some issue with the
different emission spectra of the fluorophores you are investigating and
the InSpeck beads if you use these.  However, if you are not observing
and spectral shifts and you are not at saturation in excitation then
these differences should be acceptable and the proportionality should
stay the same.

If these were my experiments I would set up a good positive control with
an standard off the shelf fluorescent probe to ensure everything is
running smoothly with each experiment.

I hope this helps.

Cheers

Steve Hunter

-----Original Message-----
From: Maria Mazzillo [mailto:[hidden email]]
Sent: Friday, 18 July 2008 11:26 PM
To: [hidden email]
Subject: Quantifying fluorescence help

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my
thesis work.  I am
looking for a reliable method for quantifying fluorescence.  

My project deals with marine dinoflagellates (zooxanthellae) that reside
intracellularly in
hosts (usually cnidarians).  I am working with cultures or isolates of
only the dinoflagellates
for my confocal work.  I have an antibody that was created against the
surface secretions
of mucilage (secreted as part of a daily cycle by the alga) from one
strain of zooxanthellae.  
I am attempting to use this antibody to label various strains to
identify differences in
mucilage between them.  Thus far, I have seen that the strain the
antibody was created
against labels around the cell fairly brightly.  Most samples either
show this or a complete
lack of labeling.  However, a few samples show a faint fluorescence
lifted off the cell
surface.  I would like to be able to quantify this fluorescence in
comparison to either the
control strain that the antibody was created against or against a known
fluorescence.  

Any help on ideas for this, or places to look for ideas would be greatly
appreciated.

Thanks!

Maria Mazzillo

[George McNamara]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Hi Maria,

See current issue of JCB:

Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy

Jiefei Geng, Misuzu Baba, Usha Nair, and Daniel J. Klionsky

J. Cell Biol. 2008 182: 129-140. Published Jul 14 2008, 10.1083/jcb.200711112. [Abstract] [Full Text] [PDF] [Supplemental Material index]  

and the Wu and Pollard 2005 paper they cite. You don't have the luxury of these yeast researchers, where they can turn up and down GFP expression like a (digital)  thermostat. I've always thought that for immunofluorescence, it ought to be possible to titrate down the immunofluorescence by mixing identical fluorophore conjugated and unconjugated antibody in appropriate amounts (i.e. 1:1, 1:3, 3:1, etc). Even better, nowadays with the right dye, such as Cy5.5 or (to help Mike I. afford college), Alexa Fluor 700, and compare results by flow cytometry, fluorescence (confocal) microscopy, microplate reader and li-Cor Odyssey quantitative Western blot scanner.

Some suggestions from me:

* as noted by others, read Pawley's "39" article in Biotechiques
* use as low magnification (but decent numerical aperture, for good light throughput) lens. low mag => more cells per field of view. More cells => higher N. "N is good".
      Decent NA means fewer optical sections to obtain complete Z-series (ideal might be one optical slice).
* Verify the instrument is producing stable results. Can be done by fluorescent beads. consider including some beads 9of comparable brightness to your cells) in with the samples. See very bottom of this message for a DIY test (or better yet, have the confocal core manager do it, so it is their dime, not yours - get the results from several nights in a row). If the lasers are not stable, your results will not be.
* consider centrifuging (lightly) the cells onto the slide, and/or other methods, so they are all in the same orientation (not some lying down, others nose/tail up - I'm much better with my coral fluorescent protein anatomy and taxonomy than for zooxanthellae: do dino's have noses?).
* these are fixed cells, right? If so, refractive index match the mounting and immersion medium, i.e. 25x/0.8 NA IMM lens with immersion oil and 2,2'-thiodiethanol (both RI 1.515, I consider 25x lens a moderate mag). With respect to TDE, read the Staudt, Hell et al 2007 Microsc Res Tech article and take the time to understand what the "ocean of fluorescence" vs RI and depth graph means for your study (hint: a perfect confocal microscope and specimen would produce a horizontal line at 1.0 relative brightness).
* if using multiple wavelengths, adjust the pinhole size(s) so that the same optical slice size is being acquired at every fluorescent wavelength. For example, if "red' 1.0 Airy unit is a 1.0 um optical slice size, set the "green" channel optical slice size to 1.0 um as well.
* You get for free the shape of the cell (and all the membranes inside it) by adding a reflected light channel. This may be helpful for segmentation (or maybe not). Reflection mode also lets you find the coverglass (very bright!) and slide (bright) surfaces. You can use 1.0 Airy units for reflection mode, as long as you are not trying to "colocalize" with the "matched thickness" fluorescence channels.
* I usually acquire Z-series with a physical step size either (a) 1/2 optical slice thickness ("overlap mode"), if bright specimen with minimal photobleaching, or (b) equal to optical slice thickness (if bleaching or on my dime). Nyquist purists would argue for 1/2.3 or even 1/3 of optical slice thickness, but then Nyquist did not have specimens that photobleached.
As Pawley wrote, "deconvolve everything" (to which I'll add; with the right algorithm, with a well functioning instrument, and verify that the output makes sense).


More interesting references:

Kai K, Kitajima Y, Hiraki M, Satoh S, Tanaka M, Nakafusa Y, Tokunaga O, Miyazaki K.
Quantitative double-fluorescence immunohistochemistry (qDFIHC), a novel technology to assess protein expression: a pilot study analyzing 5-FU sensitive markers thymidylate synthase, dihydropyrimidine dehydrogenase and orotate phosphoribosyl transferases in gastric cancer tissue specimens. Cancer Lett. 2007 Dec 8;258(1):45-54. PMID: 17892912  (in my opinion: not perfect [what is] but not bad).

Swedlow JR, Hu K, Andrews PD, Roos DS, Murray JM.
Measuring tubulin content in Toxoplasma gondii: a comparison of laser-scanning confocal and wide-field fluorescence microscopy.
Proc Natl Acad Sci U S A. 2002 Feb 19;99(4):2014-9. PMID: 11830634 (experienced microscopists, but how hard did they try to get confocal to work well? also, I do not recall them deconvolving [with a confocal optimized algorithm] the confocal data).


best wishes,

George



At 09:25 AM 7/18/2008, you wrote:

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

I am a PhD student at Auburn University just getting started into my thesis work.  I am
looking for a reliable method for quantifying fluorescence. 

My project deals with marine dinoflagellates (zooxanthellae) that reside intracellularly in
hosts (usually cnidarians).  I am working with cultures or isolates of only the dinoflagellates
for my confocal work.  I have an antibody that was created against the surface secretions
of mucilage (secreted as part of a daily cycle by the alga) from one strain of zooxanthellae. 
I am attempting to use this antibody to label various strains to identify differences in
mucilage between them.  Thus far, I have seen that the strain the antibody was created
against labels around the cell fairly brightly.  Most samples either show this or a complete
lack of labeling.  However, a few samples show a faint fluorescence lifted off the cell
surface.  I would like to be able to quantify this fluorescence in comparison to either the
control strain that the antibody was created against or against a known fluorescence. 

Any help on ideas for this, or places to look for ideas would be greatly appreciated.

Thanks!

Maria Mazzillo

... [show rest of quote]

 

George McNamara, Ph.D.
University of Miami, Miller School of Medicine
Image Core
Miami, FL 33010
[hidden email]
[hidden email]
305-243-8436 office
http://home.earthlink.net/~pubspectra/
http://home.earthlink.net/~geomcnamara/
http://www.sylvester.org/research/SR_lab_analytical.asp?ana=desc (Analytical Imaging Core Facility)

simple laser performance test to propose:

Zeiss LSM510 jargon (ChD is transmitted light detector)

4-tracks:
633 nm -> ChD
543 nm -> ChD 
488 nm -> ChD
364 nm -> ChD
(laser wavelengths can be substituted, but these are the four major channels used for immunofluorescence, so makes sense to test these. The old "LSM" software is limited to 4 tracks, 8 channels).

Use same gain setting for all ChD tracks (ex. 260 gain, 0 offset, making sure no under or over saturation). Adjust laser power to get on scale (aim for between 2000-3000 gray level to start with). Recommendation: 1.0 or 2.0 OD filter above condenser (for an inverted microscope). I leave my 1.0 OD filter taped down on the condenser dovetail, just below where the condenser polarizer swings in place.

512x512 pixel mode, 12-bit (I forget what scan speed I use, it is probably the "default").

No specimen in place (since having one could result in focus drift leading to change in brightness)

10x/0.3 NA lens is what I use. Adjust condenser focus so condenser field aperture diaphragm (CFAD) is in focus (i.e. Kohler illumination, albeit, no specimen). Adjust CFAD so it is centered and spans ~90% of the field of view (this way the corners give you the 'dark current").

Turn on the entire system (i.e. monday AM), get all this configured (i.e. "Reuse" button from a previous session), do timelapse at 1 minute interval (until first user of the week). After last user is done, run overnight. If you are (un)lucky, no user for first day, which is the best way to see how long the lasers take to reach stable operating condition.  If the facility is busy, turn off the lasers for 1 hour Friday afternoon, turn on, start immediately, run over the weekend (warning: on my LSM system, it often has problems correctly saving 2000+ timepoint series). At end (or at a break between users) I place a large circular ROI in the illuminated area (avoiding the dimmer ring of the CFAD edge), save the

Expected results:
1. good: "rock solid" (less than 10% change from min to max, and no sudden step changes) if the lasers have been on for at least 24 hours. I run mine Monday AM to Friday night (or for multiple weeks, if users have told me they might use the system over the weekend and heavy rains [hurricane] not expected). I do have a service contract with Zeiss.
2. bad: if your instrument overheats (ex. no one has ever removed the dust from the bottom of the real time computer of the LSM510 [the solution to major instabilities during the first several months of my managing this unit, until field service engineer #3 did a much more thorough job and de-dusted the RTC], and/or you have a crappy AOTF), you will see ugly changes in power in any or all wavelengths.

Note: my 364 nm line takes about 8-24 hours to reach "stable" operating condition. It always produces ugly lines in the ChD test (and DIC is ugly). Fluorescence of DAPI looks fine.


Some folks at Zeiss used to suggest that ChD detector do not report comparable intensities to the fluorescence/reflection PMTs. I have performed similar test through a fluorescent Chroma plastic slide (focused in plastic and relatively open pinhole to avoid potential focus drift "issue"), the ChD and Ch# channels track perfectly.