Calculating the pixel sizes on images

In most microscopy images that are published in research papers, there appears a scale bar.  The scale bar is like a ruler that allows you to compare sizes and distances in images from different sources.  Although a scale bar is helpful for assessing by eye, many image processing programs allows you to measure distances in the image. The problem is that these measurements are in pixels. That is what I encountered when I wanted to measure certain objects in my images. How to convert from pixels to nanometers (or microns) requires a simple formula and some prior data as follows:

  1. Objective magnification
  2. Lens magnification (in some microscopes, it is possible to get extra mag of 1.25x, 1.6x or 2x.
  3. C mount (is usually 1x)
  4. Pixel size – is the actual pixel size of the camera that is attached to the microscope.
  5. Binning – i.e. combining a cluster of pixels to a single pixel. The common options are 1X1, 2X2 and 4X4. Binning is usually used to reduce noise, but at the expense of resolution.

The formula:

Image pixel size = camera pixel size x binning / (obj. mag x lens mag x C mount)

Example:

For Cascade 512 camera (16µm/pixel on CCD), at 60x mag and 1×1 binning:

Pixel size = 16×1/(60x1x1) = 0.2667µm = 266.7nm.

Obviously, the smaller the pixel size of the camera, the better the resolution (i.e. actual pixel size in the image).

Here is a helpful table of pixel sizes (in nanometers) for some common cameras:

pixel sizes

Note that all cameras listed here have a square pixel size (e.g. 9300×9300). Some cameras have rectangular pixel sizes (e.g. ExwaveHAD 3CCD with 6350×7400).  In such cases, the length and width of the pixel sizes should be calculated separately. However, I am told that microscope-intended cameras today have only square pixels, not rectangular.

For an explanation how to add a scale bar in ImageJ, click here.

For an explanation how to add a scale bar in photoshop, click here.

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10 responses to “Calculating the pixel sizes on images

  1. Reblogged this on Science Tech Blog and commented:
    Great post on pixel size in digital microscope images

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  2. Reblogged this on The Cell Division Lab and commented:
    Great post and great blog on all things relating to fluorescent microscopy.

    Like

  3. Remember not to confuse the digital image spatial resolution (calculated a
    Pin the table above) with the optical spatial resolution (the limit of the resolution in the image info coming from the object throughg the lens and into the sensor) Abbe told us optical resolution is roughly: light wavelength / 2.NA. Where NA is the numerical aperture of the objective lens. The second number, coming after the slash after the magnification x number, written on the lens. Also remember that if the optical resolution is more than about 3x larger in nanometers than the digital image resolution then you have “empty magnification” where you don’t see any more fine details, just closer together image pixels. In this case bin the camera. In the opposite case: if the optical resolution is about the same or larger than the digital image resolution then you need more magnification, closer spaced pixels in the digital image, so resolve all the image detail the objective lens can pass through itself. See the famous article by Alvy Ray… A pixel is not a little square. Understanding this will help you not to make errors when resizing images, see here. Fiji.sc/downsample Also see teaching material at biodip.de

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  4. Is this a reliable source of information that I can cite, Does this formula really work to find the pathogen size from an image

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  5. This is very helpful. Thank you for posting. Have you got some references where you or others have used formula has been used?

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    • Thanks Prisca. I don’t have any ref. This is info I gathered online (e.g. from the sites I linked to in the post).

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      • @galicagfb
        Thank you for your reply.
        You have listed many links; which one of them exactly suggests this formula? I would like to use this formula but need to have a valid reference. do you know if that formula have been peer-reviewed?

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        • Hi Prisca,
          This is a physical propery of the camera and the calculation is just very simple math. If you wish to get a formal reference, then I suggest to look at microscope camera manufacturer websites.

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  6. Hello,
    Is there any equation or method or technique that we can calculate and find different pixel size in an image?
    Suppose we have an image and got by the camera, the camera has deformer effect on the image it means we have a lot of pixels in the image but the size is different so therefore, any method to know the size and calculate them? or make the pixel size all the same?

    Thank you very much!

    Like

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