Pixels to squared microns

[marcosfelipeti]

Hi!

I started using imageJ a few months ago, so I am not an expert on it yet. My doubt is: I have a quantity of images which express cells. I used the default settings on imageJ to calculate the surface area of these cells and then I figured out that I should have measured these in squared microns instead squared pixels. I noticed that I have to calibrate imageJ to give me this information. Does someone know how I can calibrate the scale and use the same setting for all the images that I have? (Remembering my images have different resolutions),

Thanks!

[Pariksheet Nanda]

Hi Marcos,

On Tue, Aug 12, 2014 at 12:34 PM, marcosfelipeti
<[hidden email]> wrote:
> I noticed that I have to calibrate imageJ to give me
> this information. Does someone know how I can calibrate the scale and use
> the same setting for all the images that I have? (Remembering my images have
> different resolutions),

Your image pixel calibrations will depend on the microscope you used
to capture them.

Did you use your own microscope, or someone else's (e.g. a core
facility microscope) to collect the images?  If this was someone
elses's microscope there are a few places the calibration may be
recorded:

1) Sometimes core facilities will use a stage micrometer and make a
chart of all their pixel calibrations for their objectives / cameras.
There may be a chart on the wall, or the information may be saved into
the image acquisition software and, thus, saved into your image file.
You can ask the person who maintains the microscope or software where
the calibration is recorded.

2) One can also calculate the pixel calibration reasonably well as follows:

   Camera pixel size in microns / Product of all magnifications.

Magnifications will your objective size, camera adapter magnification
(usually there is no magnification), optovar in the micoscope, etc.
For example, if you were using a camera with square 6.5 micron pixels,
a 40x objective and a 1.0x camera adapter and a 1.6x optovar, your
pixel size would be:

   6.5 microns / (40 x 1 x 1.6) = 101.6 nanometers.

Once you know the calibration you can add it to the ImageJ Image Info.


Pariksheet

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[marcosfelipeti]

Hi Pariksheet,

Thank you very much for answering! I found out that the pictures I have are in PNG format, so I converted them to TIFF format and now I can see some information. See an image sample below:

Dimensions: 498 x 109
Horizontal and Vertical resolution: 90dpi
Bit depth: 32

Online found: To convert dpi to microns: 25,400/dpi = microns, that is, 25,400/90dpi = 282.2 um

I also found : 90 dot/inch  =  0.0035433071 pixel/micrometer

I just don't know the values that I should use to calibrate the image for all the images, since all of them have 90dpi.

Thank you.

[Glen MacDonald-2]

Dear Marcos,
dpi refers to mapping the image intensities to screen location and is irrelevant to your question. Many image formats do not record spatial calibration unless the capture software writes it into the file in a manner that it can be interpreted by other applications.  Which is rarely the case with PNG and TIFF.  

You need to buy or borrow a stage micrometer and collect an image of it with each objective. the stage micrometer is a 1 mm or 10 mm long ruler etched on glass, usually available marked in 100 um, 50 um, and 10 um intervals,  and mounted on a microscope slide format.  Open the images in FIJI and The ImageJ documentation will have details on using the Set Scale command to calculate pixels/micron, or microns/pixel.  http://imagej.nih.gov/ij/docs/guide/146-30.html#toc-Subsection-30.8

Print the scale for each lens on that microscope and tape the list to the wall behind it so everyone can include the values in their notes.  Use Image>Properties to set the scale for images as you open them.

 
Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Aug 13, 2014, at 12:56 PM, marcosfelipeti <[hidden email]> wrote:

> Hi Pariksheet,
>
> Thank you very much for answering! I found out that the pictures I have are
> in PNG format, so I converted them to TIFF format and now I can see some
> information. See an image sample below:
>
> Dimensions: 498 x 109
> Horizontal and Vertical resolution: 90dpi
> Bit depth: 32
>
> Online found: To convert dpi to microns: 25,400/dpi = microns, that is,
> 25,400/90dpi = 282.2 um
>
> I also found : 90 dot/inch  =  0.0035433071 pixel/micrometer
>
> I just don't know the values that I should use to calibrate the image for
> all the images, since all of them have 90dpi.
>
> Thank you.
>
>
>
> -----
> Marcos Felipe Martins Silva
> Krasnow Institute for Advanced Study, GMU
> --
> View this message in context: http://imagej.1557.x6.nabble.com/Pixels-to-squared-microns-tp5009142p5009187.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[marcosfelipeti]

Dear Glen,

Thank you so much for replying my post! I found out that the images I download are in a scale of 105um. I am going to get my results and I hope them work out!

More once, thank you so much!

[Glen MacDonald-2]

Is that 105 um image width or 105 um per pixel?


Glen MacDonald
        Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
        Cellular Morphology Core
Center on Human Development and Disability
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]







On Aug 14, 2014, at 5:09 PM, marcosfelipeti <[hidden email]> wrote:

> Dear Glen,
>
> Thank you so much for replying my post! I found out that the images I
> download are in a scale of 105um. I am going to get my results and I hope
> them work out!
>
> More once, thank you so much!
>
>
>
> -----
> Marcos Felipe Martins Silva
> Krasnow Institute for Advanced Study, GMU
> --
> View this message in context: http://imagej.1557.x6.nabble.com/Pixels-to-squared-microns-tp5009142p5009226.html
> Sent from the ImageJ mailing list archive at Nabble.com.
>
> --
> ImageJ mailing list: http://imagej.nih.gov/ij/list.html

--
ImageJ mailing list: http://imagej.nih.gov/ij/list.html

[marcosfelipeti]

It's 105 um width and height!