Calculating Pixel Sizes and Drawing Magnification Bars

Knowing the relative size of each pixel in a digital image is extremely important. For standard electron microscopy this value is used to accurately produce a magnification bar or to measure dimensions in objects. In 3D CryoEM it is also essential for determining the CTF, calculating the reconstruction and for a wide range of other functions.

There are a number of ways of determining pixel size as well as magnification. If you have saved your CCD image in the Gatan proprietary format, DM3, then you can get this information from the Digital Micrograph program.

If you did not save your images in the DM3 format, or if you recorded them on film, then you need to know the magnification at which you recorded the image.

Take care when deciding the magnification of your image. The Tecnai operating system reports a different magnification depending on if the large viewing screen is up or down. Images recorded on the CCD camera are at a DIFFERENT magnification than those recorded with the film camera (Screen up). The easiest way to determine pixel size for an image whose magnification you know is to check the tables posted on the page Microscope magnifications and pixel sizes. It is also a good idea to know how to do the calculations without relying on the table.

Calculation of Pixel Size From Film

In this laboratory EM film is usually scanned with the Nikon Super Coolscan 8000ED at 4000 dpi.
There are 25,400 microns/inch. 25,000 microns/inch divided by the number of dpi equals the scan step size. At 4000 dpi the step size is 6.35 microns.
6.35 microns is 63,500 Å. Divide that by your magnification to get the pixel size. For example: 63,500 divided by 59,000X = 1.08 Å/pixel.

Calculation of Pixel Size From a CCD Image

You have to know the actual pixel size of the CCD cameras. The Sphera camera has 14 micron pixels and the Polara camera has 15 micron pixels.
As an example: 14 microns = 140,000 Å. You also have to know magnification of your image. Careful: The magnification on the CCD and on the film camera are different. In Digital Micrograph go to the menu Microscope/Mag Table/Edit. You can also go to the page Microscope magnifications and pixel sizes. Divide the camera pixel size in Å by the magnification to get the pixel size at the specimen level.

Adding a Magnification Bar to your Image in Adobe Photoshop

You have to know the actual pixel size of your image. Be careful that you haven't resized and resampled your image. That will change the pixel size.
Make sure the Info window is open in Photoshop (menu item Window, check Info). In the upper right corner of the Info window click on the palette options symbol and choose pixels as the unit.
If for example you wanted a magnification bar that was 500 Å long and your pixel size was, for example 1.0 Å/pixel, you would draw a line and watch in the Info window until it showed 500 pixels long.