Gain variation effects and correction in CCD cameras for TEM

By Bill Mollon, Gatan

As technology evolves, more and more people are taking the course towards replacing conventional film with the newer CCD cameras created for TEM applications. With this progression towards CCD camera use on TEM’s, is the need to fully understand all the parameters that can affect the final image quality. Gain normalization is a common procedure to correct for any artificial defects from the CCD camera and has greatly improved the image quality. However, little attention has been paid to the dependence of the gain reference image on TEM magnification.

The purpose of this article is to first demonstrate such dependence and show how it can affect the image quality. Then we show a new patented procedure to make gain reference image TEM magnification dependent.

Digital Image components

A digital image acquired from a microscope, camera, or other optical device is often described as the raw image prior to any processing or adjustment of critical pixel values (see Figure 1). In many cases, this raw image is unsuitable for use in target applications (printing, web display, reports, etc.), due to it exhibiting a significant level of noise and other artifacts from the capture system. Typical examples are detector irregularities (pixel non-uniformity and fixed-pattern contrast from the scintillator), dust, scratches, etc.. These “errors” in the raw image are manifested as dark shadows, excessively bright highlights, specks, and mottles that alter the true pixel values. Applying the gain normalization technique to raw digital images can often ensure photometric accuracy and remove common image defects to restore the fidelity of features and achieve a more visual balance (Figure 2).

Figure 1 Raw image	Figure 2 Corrected image (gain normalized)

Gain Variations Due to TEM Magnification (Beam Profile)

Successful gain normalization assumes that uniform illumination condition exist on the CCD scintillator and the gain reference image is recorded under such condition. If there is a deviation from the uniform illumination as a result of changing TEM magnification, gain normalization will certainly fail.

For bottom mounted CCD cameras, since the field of view is small compared to that of film, the variation of illumination with TEM magnification is found to be very small and can be ignored. However, for CCD cameras mounted in the 35mm port (wide angle position), such variation can not be ignored. The reason is simply that the field of view of the CCD camera is now larger than that of film. As a result, distortions in the TEM electron-optical system can significantly degrade the image quality since we can not assume the uniform illumination remains when TEM magnification is changed.

Figure 3 shows a perfect gain normalized image (no sample) just after gain reference image is recorded at 8500x using parallel illumination. The image is flat and free from any artifacts.

Figure 3 Gain normalized image of the parallel electron beam (8500x)

Using the line profile tool on this image you can see that it shows a uniform “flat” intensity response after gain normalization (Figure 4)

Figure 4 Line profile of Figure 3

Now we can show the variation of the TEM beam profile as TEM magnification changes. This variation is most pronounced when the camera is mounted in the 35mm port of the TEM due to large field of view.

The problem is noticeable if only one gain reference series is collected and is attempted to be used for all magnifications. To demonstrate this phenomenon we will change the TEM magnification and re-acquire the gain normalized image to see if there are noticeable affects on the image quality.

Magnification Affects on TEM Beam Profile

So far we have only collected on set of reference images at a magnification of 8500x. After the TEM magnification was raised from 8500x to 38,000x. A gain normalized image of the beam (using gain reference image recorded at 8500x) was then acquired. As you can see in Figure 5, the gain reference images recorded at 8500x have failed to reproduce a uniform image at this higher magnification.

Remember that we only changed the TEM magnification and nothing else. So the unevenness in the image must come from change in illumination pattern and nothing else! This has clearly demonstrated that the illumination pattern changes with TEM magnification and hence we must consider this fact when preparing gain reference images.

Figure 5 Gain normalized image of electron beam (38,000x)

If we continue our investigation and go to an even higher magnification (105,000x) you will see that the TEM beam profile has changed again resulting in another example of the reference images unable to correct properly.

Figure 6 Image of electron beam at higher magnification (105Kx)

When this phenomenon occurs and if we can store only one set of gain reference images, the end user would have to take time to properly acquire a new gain reference image in order to continue on with collecting artifact free images. This could be time consuming or even worse, overlooked until later when closely working with the acquired images. We have noticed an increase in people reporting this problem as more people enter the digital world in electron microscopy. Usually it is the Life Science applications that are most sensitive to this variance due to image acquisitions covering wide ranges in magnification and the use of wide angle CCD cameras.

For illustration purposes the examples shown here used large changes in magnification. There are cases in which a single set of reference images are adequate if the magnification step change is very small and close to the original starting magnification. As can be expected, the variance due to beam profile changes will be different for each TEM and operating condition used.

Multiple Gain Reference

Now that you have a better understanding of gain reference processing, it would be ideal if we could collect multiple sets of reference images and assign them to magnification ranges or even individual magnification steps. As the user changes magnifications the correct gain reference assigned to that magnification would be used for the acquisition. In the new Gatan Microscopy Suite, GMS 1.6 we have implemented exactly this behavior. The user now has the ability to prepare more than one set of gain reference images and assign them to individual magnification steps or even ranges. This gives the user total freedom in changing from one magnification to another without having to stop and reacquire reference images. Productivity, ease of use and controlled high quality image collection becomes the new standard in the digital acquisition session. Let’s go through the steps and use this new feature:

Assuming that GMS 1.6 has been installed the steps to prepare gain reference images has not changed that much from previous versions. The noticeable difference is the assigning of magnification ranges to the reference set.

1 If your camera requires it, prepare for collecting dark reference images first. Make sure the electron beam is off or blanked and the TEM viewing chamber is covered to prevent stray room light. Pull down the Camera menu and select “prepare dark reference”. The software will begin the process of collecting the dark reference images. 2 Next are the gain reference images. Turn the electron beam back on and make sure the specimen is removed from the beam path. Select the magnification desired for final image acquisition. 3 With the specimen removed, spread the illumination so that it is uniformly spread across the CCD and the proper number of counts per pixel is seen. Use the Intensity bar as shown in DigitalMicrograph to assist you with this (Figure 12) 4 Pull down the Camera menu and select “prepare gain reference”. You will be prompted to enter your camera’s target intensity and the magnification range you want this to be applied to (Figure 13). If you want to apply this to a single magnification, enter the same number in both boxes. 5 A dialog window will appear reminding you to make sure the illumination is uniform and that the specimen is removed. 6 During the reference image acquisition, if the software finds the illumination to the CCD is too high or too low, you will be prompted to adjust the intensity.

Figure 12

Summary

Depending on your TEM model you may find that several successive magnification steps do not change the beam profile that much. Most TEM’s have at least 3 basic operating magnification ranges: low (1000x – 10,000x), medium (11,000x – 40,000x) and high (50,000x – 300,000x). In order to find out how changing the magnification affects the gain reference images in these ranges you can start at the low end and collect the first set. Then with gain normalization processing turned on, collect an image of the beam after increasing the magnification. Look for affects shown in Figures 10 and 11. When you notice an appreciable change, record the magnification step and collect a new gain reference set there. This should help you “see” the magnification ranges on your TEM where a gain reference set should be collected and be used later in correcting your final image.

Once this is completed you should not have to recollect gain reference images unless you have changed other illumination or optical parameters on your TEM such as condenser aperture size, objective aperture size, accelerating voltage and in some cases spot size. Overall illumination intensity delivered to the CCD camera by the TEM optics will have an affect on the gain reference images collected. Keep in mind that gain reference images are actually a series of snapshots of your current beam operating conditions that you have set on the microscope. Dark reference images should be re-acquired periodically as a maintenance check or if instructed by the Gatan Service engineer as part of troubleshooting. As mentioned earlier the dark reference images correct for components of the CCD and camera electronics and are independent of TEM beam illumination.

The development and investigation of this technique came from valuable input that we receive from our customers. This is a perfect example of the commitment that we take to help improve our products based on the needs of our customers. We hope you find this information and new software feature gives you assurance in collecting quality, consistent, easy to use and reliable digital images from our cameras. Allowing you to spend more time on your application and letting our systems deliver a solution is our goal.

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