Question about Optical Density

[Francisco J H Blazquez]

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Good morning


I have a question about ImageJ measurement of optical density in photomicrographs obtained by digital cameras. As I understand the image captured by the camera CCD register the transmitted light. That should mean that the voltage (analogical signals) produced by the CCD is proportional to the brightness of the object. After the conversion of these signals to digital information, the brightness and gray shades of the object are translated into pixels values of gray that goes from 0 (black) to 255 (white).


If a pixel(a) has a gray value of 100 when compared with another pixel(b) that has a gray value of 200 we may conclude that the first one (a) is darker than the second one (b) and we may conclude that the object region corresponding to the first pixel with the gray value of 100 is also darker.
So, when we take a measurement of the mean gray level of an area of an object (translated as Optical Density – OD), be it by using ImageJ or Axiovision (Zeiss), we should conclude that the higher values correspond to a general bright object area and the lower values correspond to a darker area of the object. In other words the value of OD has an inverse correlation with the “darkness” of the area measured.
That reasoning may be extended to the Integrated Optical Density (IOD) that is calculated multiplying the pixel mean gray value of the area by the area of the region of the object where the measurement was taken (OD)Xarea = IOD, IOD is also the sum of the gray values of the area. That is, the lower the IOD number, the darker is the Area.


If we are measuring the product of an enzyme histochemical reaction or DAB deposition in a immunohistochemical DAB reaction, we may conclude that the lower the IOD, the darker is the area and consequently, the more quantity of reaction product is present the area. Of course, this relationship must be inverted if the image was originated from immunofluorescence.


Am I right? Is there something that I am not considering?

Francisco Blazquez
School of Veterinary Medicine
University of Sao Paulo

[Kevin Ryan]

You have the basics correct. The optical density measurement is one of absorbance, which can be expressed as the log of transmittance:

    OD = -log(Transmittance) = -log(Intensity of a blank / Intensity of the specimen)

A transmittance of 1.0 indicates no absorbance, and thus an OD of 0.0. From the ever handy WikiPedia (https://en.wikipedia.org/wiki/Absorbance):

Absorbance: −log10(Φet/Φei) Transmittance: Φet/Φei)
        0 1
        0.1 0.79
        0.25 0.56
        0.5 0.32
        0.75 0.18
        0.9 0.13
        1 0.1
        2 0.01
        3 0.001

High OD values are, unfortunately, less certain - there's far less grayscale change involved in 2.0->2.1 OD than there is in 0.0->0.1 OD. A transmittance of 0.0 indicates an infinite OD as a result. This means that if you are quantifying OD you want _some_ transmittance in even the darkest portions of the sample, if at all possible.

Most imaging packages can apply an intensity calibration to convert grayscale to optical density/absorbance in transmitted light microscopy, with the OD scale indicating in a linear fashion the amount of material present. Note that in many packages (for example Image-Pro Plus/Premier, which I'm quite familiar with as our products) apply the current intensity calibration to _whatever_ is being measured, so that if an OD calibration has been applied to an image the IOD measurement is in OD terms, not the original gray levels. Check your software documentation on this to be certain that this is the case for your system.


Kevin Ryan
Senior Project Manager
Media Cybernetics, Inc.


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of [hidden email]
Sent: Friday, August 21, 2015 9:00 AM
To: [hidden email]
Subject: Question about Optical Density

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Good morning


I have a question about ImageJ measurement of optical density in photomicrographs obtained by digital cameras. As I understand the image captured by the camera CCD register the transmitted light. That should mean that the voltage (analogical signals) produced by the CCD is proportional to the brightness of the object. After the conversion of these signals to digital information, the brightness and gray shades of the object are translated into pixels values of gray that goes from 0 (black) to 255 (white).


If a pixel(a) has a gray value of 100 when compared with another pixel(b) that has a gray value of 200 we may conclude that the first one (a) is darker than the second one (b) and we may conclude that the object region corresponding to the first pixel with the gray value of 100 is also darker.
So, when we take a measurement of the mean gray level of an area of an object (translated as Optical Density – OD), be it by using ImageJ or Axiovision (Zeiss), we should conclude that the higher values correspond to a general bright object area and the lower values correspond to a darker area of the object. In other words the value of OD has an inverse correlation with the “darkness” of the area measured.
That reasoning may be extended to the Integrated Optical Density (IOD) that is calculated multiplying the pixel mean gray value of the area by the area of the region of the object where the measurement was taken (OD)Xarea = IOD, IOD is also the sum of the gray values of the area. That is, the lower the IOD number, the darker is the Area.


If we are measuring the product of an enzyme histochemical reaction or DAB deposition in a immunohistochemical DAB reaction, we may conclude that the lower the IOD, the darker is the area and consequently, the more quantity of reaction product is present the area. Of course, this relationship must be inverted if the image was originated from immunofluorescence.


Am I right? Is there something that I am not considering?

Francisco Blazquez
School of Veterinary Medicine
University of Sao Paulo

[Sathya]

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Hi  Francisco,
If you are considering DAB staining for quantification of intensity, do consider these discussions about the DAB quantification:
 
- https://list.nih.gov/cgi-bin/wa.exe?A2=ind0902&L=IMAGEJ&P=R18412
 
- http://imagej.1557.x6.nabble.com/DAB-quantification-td5006159.html
 
There are some inherent problems while using DAB for quantification. Good luck.
 
Sathya Srinivasan
Manager
RUN Advanced Optical Microscopy Core Facility
(www.ucalgary.ca/runcore)
University of Calgary
Calgary, AB T2N4N1
Canada
 

> Date: Fri, 21 Aug 2015 09:59:54 -0300
> From: [hidden email]
> Subject: Question about Optical Density
> To: [hidden email]
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
>
> Good morning
>
>
> I have a question about ImageJ measurement of optical density in photomicrographs obtained by digital cameras. As I understand the image captured by the camera CCD register the transmitted light. That should mean that the voltage (analogical signals) produced by the CCD is proportional to the brightness of the object. After the conversion of these signals to digital information, the brightness and gray shades of the object are translated into pixels values of gray that goes from 0 (black) to 255 (white).
>
>
> If a pixel(a) has a gray value of 100 when compared with another pixel(b) that has a gray value of 200 we may conclude that the first one (a) is darker than the second one (b) and we may conclude that the object region corresponding to the first pixel with the gray value of 100 is also darker.
> So, when we take a measurement of the mean gray level of an area of an object (translated as Optical Density – OD), be it by using ImageJ or Axiovision (Zeiss), we should conclude that the higher values correspond to a general bright object area and the lower values correspond to a darker area of the object. In other words the value of OD has an inverse correlation with the “darkness” of the area measured.
> That reasoning may be extended to the Integrated Optical Density (IOD) that is calculated multiplying the pixel mean gray value of the area by the area of the region of the object where the measurement was taken (OD)Xarea = IOD, IOD is also the sum of the gray values of the area. That is, the lower the IOD number, the darker is the Area.
>
>
> If we are measuring the product of an enzyme histochemical reaction or DAB deposition in a immunohistochemical DAB reaction, we may conclude that the lower the IOD, the darker is the area and consequently, the more quantity of reaction product is present the area. Of course, this relationship must be inverted if the image was originated from immunofluorescence.
>
>
> Am I right? Is there something that I am not considering?
>
> Francisco Blazquez
> School of Veterinary Medicine
> University of Sao Paulo

     

[Francisco J H Blazquez]

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Thank you all, I am finding this discussion enormously instructive.

I am new to OD measurement and it will take me some time to digest all this information, George, but I really thank you very much for the time that you spared to share your experience.

Sincerely

> Francisco Blazquez
> School of Veterinary Medicine
> University of Sao Paulo

----- Mensagem original -----

> De: "George McNamara" <[hidden email]>
> Para: "Confocal Microscopy List" <[hidden email]>,
> [hidden email]
> Cc: "Sathya Srinivasan" <[hidden email]>
> Enviadas: Sábado, 22 de Agosto de 2015 2:43:35
> Assunto: Re: Question about Optical Density

> Hi Sathya,
> Excellent links - though a lot more redundent claims than data.

> Hi Francisco,

> The online document, " Using Optical Density (Scaled) for
> Densitometry "
> ftp://ftp.meta.moleculardevices.com/pub/uic/software/MM50/MetaMorph%20Extras/Application%20Notes/ODSCALE.DOC
> .

> if the above link does not work, try using google to search for:
> metamorph scaled optical density odscale.doc

> may be of use - even though it was written in 1997 (just a youngster
> compared to the Beer–Lambert–Bouguer law
> https://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law ).

> lot's of issues - some of which I'll even get to.DAB is not so bad -
> "talk is cheap" (as in data free electronic posts), data is better -
> more on this later (no DAB data below, but I probably have more
> experience integrating DAB and other IHC slides than all the people
> who posted on the sites Sathya mentioned)..

> Biggest point for transmitted light optical density measurements of
> small objects (small relative to the field of view): "glare this the
> killer". Good references:

> Chieco P, Jonker A, De Boer BA, Ruijter JM, Van Noorden CJ. Image
> cytometry: protocols for 2D and 3D quantification in microscopic
> images. Prog Histochem Cytochem. 2013 Jan;47(4):211-333. doi:
> 10.1016/j.proghi.2012.09.001. Epub 2012 Nov 10. Review. PubMed PMID:
> 23146330.

> Jonker A, Geerts WJ, Chieco P, Moorman AF, Lamers WH, Van Noorden CJ.
> Basic strategies for valid cytometry using image analysis. Histochem
> J. 1997 May;29(5):347-64. Review. PubMed PMID: 9184850.

> Chieco P, Jonker A, Melchiorri C, Vanni G, Van Noorden CJ. A user's
> guide for avoiding errors in absorbance image cytometry: a review
> with original
> experimental observations. Histochem J. 1994 Jan;26(1):1-19. Review.
> PubMed PMID:
> 7513318.

> the same author's RMS Handbook:

> Image Cytometry
> Author/Editor(s): P Chieco; A Jonker; Cornelis J. F. van Noorden;
> Royal Microscopical Society (Great Britain)
> ISBN: 0387916180, 9780387916187
> Publisher: Springer-Verlag
> Published Place: New York
> Published/Copyright Year: 2001
> Physical Description: Illustrations included, 24 cm
> Series: Microscopy Handbooks ( Volume 46 )
> Number of pages: 116 (x, 116 pages)
> Categories:
> Cytophotometry > Handbooks, Manuals, Etc
> Imaging Systems In Biology > Handbooks, Manuals, Etc
> Cytology > Handbooks, Manuals, Etc
> information source - http://isbnplus.com/0387916180
> see also
> https://books.google.com/books/about/Image_Cytometry.html?id=fQRtQgAACAAJ

> A major point: getting correct measurements of O.D. from a light
> microscope imaging system requires complete control of the entire
> instrument. Getting reproducible data day to day if you do not
> maintain that control - good luck.

> Another major point: under normal operating conditions in a
> biomedical research lab or light microscope core facility,
> fluorescence intensity data is not quantitative either. Getting
> within 5% is considered pretty good - see Brown et al 2015:
> Brown CM, Reilly A, Cole RW. A Quantitative Measure of Field
> Illumination. J Biomol Tech. 2015 Jul;26(2):37-44. doi:
> 10.7171/jbt.15-2602-001. PubMed PMID:
> 25802488; PubMed Central PMCID: PMC4365985.
> http://www.ncbi.nlm.nih.gov/pubmed/25802488
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365985/ (open access).
> Still don't believe me about routine fluorescence microscopy
> intensity data being unquantitative? Two simple tests:
> 1. zoom down (1x, 0.7x is even better) as much as possible on your
> confocal microscope. Place a fluorescent bead in the middle of the
> field of view. Acquire an image. Move the stage so the bead is just
> barely entirely in the field of view. Acquire an image. Measure the
> intensities of the bead in the two images (a ROI is better than
> brightest pixel - the latter breaks Borisy's Law: "do not rely on a
> single pixel").
> 2. Widefield or confocal: take your favorite immunofluorescence or
> GFP specimen, optimize everything, acquire an image. Position a nice
> bright object with dark neighborhood on the stage. Now acquire a
> timelapse series of the same field of view for 10 minutes, with
> continual illumination the entire time (ex. 1 second exposure time
> => 600 image frames). Measure the ROI, export to Excel, generate
> random numbers in the next column, select both the measurement and
> random columns, sort on the random numbers (I call this
> "anti-sort"), compare the first two numbers: are they practically
> identical? Unlikely! Or, graph the measurements. Or compute the mean
> and standard deviations of the measurements. Now, in this
> experiment, you know why the data turned out this way (unless you
> cheated and used the Chroma plastic slides or ArgoLight-M slide or
> equivalent). But in real life experiments, if you are futzing with
> the focus, or repositioning the stage, or leave to take a coffee
> break, while the excitation light is on, you are unlikely to equally
> photobleach every field of view. If you use transmitted light to
> focus, good luck getting exactly the same focus point every single
> time.

> * Do not trust the NAME of measurement parameters - especially
> ImageJ's lazily named parameters. Here are the Results from Fiji
> ImageJ's Ana;yze menu -> Measure command:

> # Area Mean Min Max IntDen RawIntDen
> 1 65536 208.544 96 255 13667167 13667167
> 2 65536 0 0 0 0 0
> 3 65536 255 255 255 16711680 16711680

> where "IntDen' is short for "Integrated density" (Analyze -> Set
> Measurements). To be clear - the ImageJ does NOT claim this is
> "optical density" (though I've seen publications that use the values
> as if they were O.D.) - help page:

> "Integrated Density - Calculates and displays two values: "IntDen"
> (the product of Area and Mean Gray Value ) and "RawIntDen" (the sum
> of the values of the pixels in the image or selection). "RawIntDen"
> is only available in ImageJ 1.44c or later. "IntDen" and "RawIntDen"
> values are the same for uncalibrated image. The Dot Blot Analysis
> example demonstrates how to use this option to analyze a dot blot
> assay."

> FYI - When I learned math, I was taught to calculate the Mean FROM
> the sum of the values divided by the number of values.

> Back to the data - the values in the first row are from the Fiji logo
> file (image folder). Second row is from a black (zero values) image,
> 3rd row is from a white (255 value pixels0 image. If this

> Back to OD -

> Putting a stack of 100 OD 1 filters (with air gaps in between) would
> enable you to create a specimen of 100 OD, good luck measuring
> accurately (other than taking it apart and counting each filter ...
> yes, I realize you wrote gray levels - ImageJ doesn't take what the
> numbes a). "Garbage in, garbage out". Reasonable expectations for
> optical densities are in the range of around 0 to 2 by eye or a
> camera under typical use. Chroma, Semrock and other filter companies
> routinely show their spectra of their interference filters as %T or
> O.D. with OD down to 6.0, sometimes beyond that (7.0 on a very good
> day). They are not using a microscope-camera setup for these
> measurements! Call up tech support for each and ask (1) how do they
> do the measurements on their filters, and (2) what do they think of
> the OD spectra reported by the other company.

> "If a pixel(a) has a gray value of 100 when compared with another
> pixel(b) that has a gray value of 200 we may conclude that the first
> one (a) is darker than the second one (b) and we may conclude that
> the object region corresponding to the first pixel with the gray
> value of 100 is also darker."

> Very carefully written! Most scientists would assume that 200 is 2x
> of 100, that is, in the case of light microscopes and cameras, 200
> is 2x as many photons as 100. If the camera offset (and dark current
> for long exposures) has been subtracted out, this is correct for
> Scientific CCD and Scientific sCMOS cameras. However, for most
> cameras, flatbed scanners, etc, which apply a Gamma to the data
> after the sensor and before the application software, 100 and 200
> are not linear with respect to photons. Most flatbed scanners can do
> a pretty good job, if "raw" data is output - for example, using
> www.hamrick.com VueScan Pro, with raw data output. Think this cannot
> happen in science, or in Science? See
> http://www.ncbi.nlm.nih.gov/pubmed/16224022 for systematically
> incorrect data from a flatbed scanner -0- which usually would not be
> a problem, except their "quantitative" fluorescence microscopy data
> is "cvalibrated" against their flatbed scanner results: "
> Fluorescence measured by microscopy or flow cytometry was directly
> proportional to protein concentration measured by quantitative
> immunoblotting. ".

> Ooops:

> "That reasoning may be extended to the Integrated Optical Density
> (IOD) that is calculated multiplying the pixel mean gray value of
> the area by the area of the region of the object where the
> measurement was taken (OD)Xarea = IOD, IOD is also the sum of the
> gray values of the area. That is, the lower the IOD number, the
> darker is the Area. "

> Incorrect! You are obtaining the "mean pixel gray value" FROM the
> integrated optical density and whatever number of pixels you have
> chosen. If you are measuring a small object, in a large field of
> view that is otherwise empty, -- and have glare controlled correctly
> (as well as practical) -- the mean will vary depending on how many
> pixels away from the object you include. The bigger the region, the
> smaller the mean value ... but this is a silly thing to do since you
> could use a small number of pixels OR the entire camera field of
> view, and calculate a mean that is for practical worth, meaningless.
> The right thing to do is to report the Integrated Optical Density of
> the object -- if the rest of the field of view is empty, all those
> pixels have individual OD. values of zero, so "adding" zeros to the
> object IOD adds nothing. More practically, the values would be so
> close to zero they should be thresholded out. A useful rule of thumb
> being that an 8-bit (linear data out!) camera has a useful pixel OD
> range of about 0.05 to 1.2 (if glare is well controlled) and a
> scientific CCD or sCMOS around 0.05 to 2.0 (maybe 3.0 if you are a
> Jonker et al caliber expert).

> DAB - not really all that bad. The stuff on an IHC slide is a POLYMER
> that scatters light. Scatters a lot at short wavelength (ex. 420
> nm), not so much as 600 nm, very little at 800 nm. Some of the
> variation between manufacturer's is that some include Nickel ions or
> Cobalt ions in their DAB formulations. Use a 420 nm narrow band
> filter is ideal for DAB on a light microscope (and scientific CCD or
> sCMOS and correct glare control). 600 nm is around the absorption
> peak of typical hematoxylin (though hematoxylins can vary too). This
> means that if you acquire the right monochromatic images, do the
> right OD image conversions and calculations, you can compute:

> OD420 - OD420(Hematoxylin) ... where this is scaled from say
> OD600(Hematoxylin) ... to get the OD420(DAB) contribution.

> I worked out most of this on a CompuCyte imaging laser scanning
> cytometer while working at City of Hope National Medical Center
> (2005-2006) - hopefully that iCyte or iCys has been retired.
> CompuCyte was bought by ThorLabs if you really want one -
> http://www.thorlabs.com/navigation.cfm?guide_id=2333
> Another instrument that might be usable (and could scan an entire
> adult human brain tissue section in one click) is Tissue Scope from
> http://www.hurondigitalpathology.com/ (be sure to pick out the laser
> lines yourself).

> It is most prudent to image the single stained controls to become
> comfortable with this. I also note a good source of monochromatic
> light are the various laser lines on a laser confocal microscope,
> with 405 nm being close enough to my preference of 420 nm (and it is
> just a preference - a relatively narrow bandpass DAPI filter would
> work, a narrow pass BV421 emission filter should be very nice).

> By the way ... Above 700 nm, few IHC reagents absorb light (since
> pathologists and histologists cannot usually see 'up there'). Many
> flatbed scanners and 35 mm film scanners can acquire a NIR channel
> image to facilitate identifying scratches and dust -- the
> Hamrick.com VueScan Pro can let you acquire those on scanners that
> have this -- see
> http://works.bepress.com/cgi/viewcontent.cgi?article=1027&context=gmcnamara

> Even simpler ways to get quantitative histolofy and IHC are to find
> -- or buy -- either:
> 1. an ASI SKY system,
> http://www.spectral-imaging.com/products-technologies/spectral-imaging
> (the Sagnac interferometer based systems, not the color camera
> ones!).
> 2. PerkinElmer/CRi nuance (the camera) or Vectra (the digital slide
> imager).
> Both have OD calculation capabilities built in (unless ASI took
> theirs out).

> Sets of O.D. filters can be purchased from several commpanies, such
> as
> Thorlabs --
> https://www.thorlabs.com/NewGroupPage9.cfm?ObjectGroup_ID=266
> Edmund Optics / Edmund Scientific
> Melles Griot / CVI / IDEX (a sister company to Semrock) --
> http://marketplace.idexop.com/store/absorptive-neutral-density-filter-sets
> Stouffer -- http://www.stouffer.net/Specialtyguides.htm -- note there
> are different step tablets for reflection and transmission.
> Some calibration instructions (not that I trust any documentation
> stat starts with "252.37 = O.D. 0.00"
> http://imagej.nih.gov/ij/docs/examples/calibration/

> you may also find of use Macville et al 2001 ...
> downloads.hindawi.com/journals/acp/2001/740909.pdf
> http://www.ncbi.nlm.nih.gov/pubmed/11455032

> There are bunch of OD spectra of IHC dyes -- and DAB -- in the XLSX
> file inside the zip file at
> http://works.bepress.com/gmcnamara/9/

> and maybe some additional links at
> http://home.earthlink.net/~pubspectra/ (after the two pictures that
> do not show up in web browsers - they do download).

> I don't have DAB&H posted online (other than inside the PubSpectra
> XLSX file), but some may find of interest the H&E spectra at
> http://home.earthlink.net/~geomcnamara/skyomics.htm
> and for more fun,
> http://home.earthlink.net/~geomcnamara/CrusadeBetterMicrographs.htm

> Enjoy,

> George

> On 8/21/2015 2:04 PM, Sathya Srinivasan wrote:
> > *****
>
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images
> > on
> > http://www.imgur.com and include the link in your posting.
>
> > *****
>

> > Hi  Francisco,
>
> > If you are considering DAB staining for quantification of
> > intensity,
> > do consider these discussions about the DAB quantification:
>
> >
>
> > - https://list.nih.gov/cgi-bin/wa.exe?A2=ind0902&L=IMAGEJ&P=R18412
> > -
> > http://imagej.1557.x6.nabble.com/DAB-quantification-td5006159.html
> > There are some inherent problems while using DAB for
> > quantification.
> > Good luck.
>
> >
>
> > Sathya Srinivasan
>
> > Manager
>
> > RUN Advanced Optical Microscopy Core Facility
>
> > ( www.ucalgary.ca/runcore )
>
> > University of Calgary
>
> > Calgary, AB T2N4N1
>
> > Canada
>
> > > Date: Fri, 21 Aug 2015 09:59:54 -0300
> >
>
> > > From: [hidden email] Subject: Question about Optical Density
> >
>
> > > To: [hidden email] *****
> >
>
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images
> > > on
> > > http://www.imgur.com and include the link in your posting.
> >
>
> > > *****
> >
>

> > > Good morning
> >
>

> > > I have a question about ImageJ measurement of optical density in
> > > photomicrographs obtained by digital cameras. As I understand the
> > > image captured by the camera CCD register the transmitted light.
> > > That should mean that the voltage (analogical signals) produced
> > > by
> > > the CCD is proportional to the brightness of the object. After
> > > the
> > > conversion of these signals to digital information, the
> > > brightness
> > > and gray shades of the object are translated into pixels values
> > > of
> > > gray that goes from 0 (black) to 255 (white).
> >
>

> > > If a pixel(a) has a gray value of 100 when compared with another
> > > pixel(b) that has a gray value of 200 we may conclude that the
> > > first
> > > one (a) is darker than the second one (b) and we may conclude
> > > that
> > > the object region corresponding to the first pixel with the gray
> > > value of 100 is also darker.
> >
>
> > > So, when we take a measurement of the mean gray level of an area
> > > of
> > > an object (translated as Optical Density – OD), be it by using
> > > ImageJ or Axiovision (Zeiss), we should conclude that the higher
> > > values correspond to a general bright object area and the lower
> > > values correspond to a darker area of the object. In other words
> > > the
> > > value of OD has an inverse correlation with the “darkness” of the
> > > area measured.
> >
>
> > > That reasoning may be extended to the Integrated Optical Density
> > > (IOD) that is calculated multiplying the pixel mean gray value of
> > > the area by the area of the region of the object where the
> > > measurement was taken (OD)Xarea = IOD, IOD is also the sum of the
> > > gray values of the area. That is, the lower the IOD number, the
> > > darker is the Area.
> >
>

> > > If we are measuring the product of an enzyme histochemical
> > > reaction
> > > or DAB deposition in a immunohistochemical DAB reaction, we may
> > > conclude that the lower the IOD, the darker is the area and
> > > consequently, the more quantity of reaction product is present
> > > the
> > > area. Of course, this relationship must be inverted if the image
> > > was
> > > originated from immunofluorescence.
> >
>

> > > Am I right? Is there something that I am not considering?
> >
>

> > > Francisco Blazquez
> >
>
> > > School of Veterinary Medicine
> >
>
> > > University of Sao Paulo
> >
>
> --

> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales http://works.bepress.com/gmcnamara/42