Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/intensity-measurements-tp3005594p3016215.html
Hi Vaishali ,
Yes, MetaMorph has the image processing command, "Optical Density
(Scaled)" [Process menu ... it is a dropin, so you may need to add
it using mmadmin], that performs both shading correction and the -log10
conversion (the scaled part is you multiple by 100 or 1000 to end up with
integer pixels values since MetaMorph does not have [nor especially
needs] a floating point image type).
specimenimage(s) - darkreference
OD(scaled) = scalingfactor * -log10 (
--------------------------------------------------- )
whitereference - darkreference
where
whitereference = blank field of view (clean area(s) on microscope slide
... ideally the median [at each pixel] from a stack of 10 or more fields
of view (the version of MetaMorph I have does not have a median operation
in Stack Arithemtic, so use Average instead after deleting planes with
ugly junk in them). Needs to be images with the slide in place, otherwise
you mess up Koehler illumination.
and
darkreference = no light to the camera ... ideally the average of 10 or
more exposures.
The inside the (------) is the standard shading correction operation. In
the ODscaled command the denominator needs to be pre-calculated
("shading reference"). Use offset=0.
Assumption: that the microscope is in Koehler illumination - especially
that the condenser NA is adjusted correctly, lenses clean, monochromatic
light (for each chromogen or dye if using two or more). The condenser NA
needs to be adjusted correctly and then left alone. Too small an NA will
result in lousy resolution and all kinds of junk showing up from out of
focus, too high a condenser NA will result in glare (upshot: do not just
push the condenser NA to fully open; glare is an OD dynamic range
killer).
For more on MetaMorph's use:
http://support.meta.moleculardevices.com/docs/
T10012 Using Optical Density (Scaled) for Densitometry [refers to
version 2.5 - quaint].
The -log10 conversion can just as easily be done by exporting gray levels
to Excel and then doing the calculation (no need for the
"scaled" then).
DAB is a scatterer, so strictly speaking, Beer-Lambert law does not apply
to it. However, with internal standards, for example, cell lines
expressing known quantities of your favorite antigen), Western blot or
ELISA, etc, you could convert to "real life" units, such as:
molecules/um^2 (ideally). I also strongly urge that you purchase a
calibrated OD standard (Edmund Scientific's "neutral density step
tablet" is fine, Reynard Corp,
http://www.reynardcorp.com/filters-step-linear-neutral-density-filters-c-1_2-l-en.html
but all these slides are 2 mm thick, or film based ones from Kodak or
Stouffer.net).
DAB IHC vs fluorescence:
The dynamic range of DAB IHC is much less than fluorescence if you are
using a scientific grade CCD camera for the detector (ex. Hamamatsu
ORCA-ER). On a good day, your dynamic range in O.D. units is going to be
about 0.05 to 1.2 (if operating in 8-bit range, which you should not do)
or 0.05 to almost 2.0 (if 12-bit CCD, no staining to almost 1%
transmission). Dynamic range of fluorescence can be huge, if your
background (from all sources, but most obvious issue is from the tissue
autofluorescence) is really low.
The following paper makes a good case for fluorescence, but the author is
cofounder (and patent co-inventor) of a fluorescence imaging method
("AQUA" score is basically an optical equivalent of unsharp
masking followed by a scaling to useful 8-bit dynamic range ... company
was too chicken to report in real number of molecules per um^ or per
cell, and the unsharp masking operation could be replaced by a much more
quantitative deconvolution algorithm with little extra acquisition or
processing time):
Rimm DL.
What brown cannot do for you. Nat Biotechnol. 2006 Aug;24(8):914-6.
Review. PMID: 16900128
Color vs monochrome camera:
While in principle Beer-Lambert law refers to monochromatic light, a
typical fluorescence bandpass filter is fine for OD imaging. Even the
color filters in the color cameras are good enough (and pixel resolution
is typically not an issue, or can use a 40x/0.75 NA lens instead of a
20x/0.75 NA lens if you want or need to).
The BAD feature about color cameras is that they typically default to
gamma NOT EQUAL to one. This is not a killer, just need to be aware of
it. This also mars most papers that use a desktop scanner for
"quantitative densitometry" of chemiluminescent Western blots
(or autoradiograms). For example, the otherwise nice papers by Wu and
Pollard doing "absolute quantitation" report absolutely
incorrect values (probably off by no more than a factor of three, which
is probably close enough for a biochemist) because they do not understand
that their cheap desktop scanner is designed to make color photographs of
people's faces show up ok on their monitor.
Chapter 9: Counting proteins in living cells by quantitative fluorescence
microscopy with internal standards.
Wu JQ, McCormick CD, Pollard TD.
Methods Cell Biol. 2008;89:253-73.
PMID: 19118678
Counting cytokinesis proteins globally and locally in fission
yeast.
Wu JQ, Pollard TD.
Science. 2005 Oct 14;310(5746):310-4.
PMID: 16224022
If you want to use your scanner as a densitometer, buy VueScan Pro and
use 16-bit TIFF output
(
http://www.hamrick.com/vuescan/html/vuesc32.htm#outputrawfile).
SilverFast Ai has a similar "raw' output option and is bundled with
some scanners.
If you have a color camera that is not linear (or a monochrome camera
that is not linear!), and invest in an OD step tablet, then you can
generate a calibration curve in Excel (or MetaMorph).
Here is my KISS color imaging method for DAB&Hematoxylin:
monochrome CCD (ex. ORCA-ER)
DAPI bandpass filter cube -> DAB image channel
Cy3 bandpass filter cube -> hematoxylin image
channel
Set up exposure time(s) so the bright background in each is in the 3000
to 3500 intensity range (on ORCA-ER the dark reference image will be
around 200, so you will have ~3000 intensity units to play with. In
theory should be ~3 OD, but reality is that no one will be able to tell
by eye a cell with average OD of 2.0 vs 3.0 on the microscope, so the
staining protocols will never be designed to do this).
For color image:
R = Cy3 filter cube image
G = Cy3 filter cube image (or, to get funky, stick in the DAPI channel
image instead)
B = DAPI filter cube image
If possible, replace hematoxylin with methyl blue (see Chantrain et al
2003 J Histochem Cytochem, note: we did NOT do OD imaging).
If you are serious about OD imaging, make extra serial sections and do
DAB only on half of them.
If you are willing to try out OD vs fluorescence, you can use fluorescent
tyramide as a direct replacement for DAB (money note: if you are not
paying attention to details and figure out how to minimize the reagent
volume, you might end up pipeting $100 of fluorescent tyramide on a
single slide). You will probably need to re-titer your antibodies. See
Brian Van Tine's methods paper for more on fluorescent
tyramides:
Simultaneous in situ detection of RNA, DNA, and protein using
tyramide-coupled immunofluorescence.
Van Tine BA, Broker TR, Chow LT.
Methods Mol Biol. 2005;292:215-30.
PMID: 15507711
Sincerely,
George
At 06:51 AM 6/1/2009, you wrote:
hello friends,
We are interested in measuring the intensity in immnohistochemical slides
(DAB stained).
Could anybody suggest some software or a method to quantitate DAB
intensity in tissue sections.
Secondly,can these measurements be made using metomorph, ImageJ or
Axiovision as these are the softwares available with us.
Thanks
Vaishali Kailaje
Scientific Assistant,
Tata Memorial Centre,
Advanced Centre for Treatment Reasearch Education in Cancer
Kahrghar,
Navi Mumbai,
India.
George McNamara, Ph.D.
Image Core Manager
Analytical Imaging Core Facility
University of Miami, Miller School of Medicine
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/AICF (Analytical Imaging Core Facility)