Calibrating Electron Dose

There are circumstances where the user of a TEM needs to have a reasonably accurate estimate of the electron dose for a given specimen or set of images or for images of the same specimen recorded over multiple sessions on the TEM. In addition, software such as Gatan's DigitalMicrograph package uses the relationship between beam current on the sample and the counts recorded in an image to estimate the electron dose that the specimen receives (or perhaps better put, to estimate the number of electrons per pixel in the image that produces its contrast). Other software such as serialEM can control TEM settings with respect to the electron beam incident on the specimen, and track the electron dose regardless of the amount of scattering that the specimen causes. All these indicators of the electron dose at the specimen require some sort of calibration of both the instrument and the software.

Numerous procedures exist to estimate the absolute strength of the electron beam in a TEM, but the most accurate way to determine and calibrate the electron dose on an electron microscope is through the use of aFaraday cup. This allows the user to measure the beam current (with nothing in the beam path) very accurately under a variety of different beam conditions (e.g., changing the condenser apertures and spot sizes) and to correlate the beam current with lens settings, camera outputs, etc.

calibration curve of counts per electron for the EMC's JEOL JEM 3200FSIn Jan, 2015, Jaap Brink (TEM product manager for Life Sciences with JEOL), Mike VanEtten (one of the JEOL service engineers for the Bloomington area) and David Morgan calibrated the electron dose on the EMC's JEOL JEM 3200FS using a Faraday cup. This involved recording the beam current using the Faraday cup while different combinations of second condenser lens aperature (CLA settings 1 through 4 - largest to smallest - in terms of the operation of this instrument) and spot size settings 1 through 5 (strongest to weakest electron beam) were used to adjust the strength of the electron beam. At the same time, an image of the actual electron beam was acquired using the UltraScan 4000 CCD camera. Imaging conditions were chosen so that all the images could be recorded under the same conditions over the range of beam current created by the various aperture/spot size combinations. For the 3200FS and the UltraScan 4000 CCD camera combination, an exposure of 0.25 s at a magnification of 30,000x was used. The electron beam was centered within the CCD frame in order to include the entire electron beam within each image. In addition, for the exposures where the electron beam was at its most intense (larger condenser aperatures and lower spot size numbers), the beam was expanded to cover as much of the CCD as possible while still being completely on the detector. This step was critical in order to not over-saturate the CCD elements and (potentially) damage the camera.

Note: Provided that the electron beam stays completely within the Faraday cup, the current reported by the device does not depend on things like lens astigmatism and the size of the electron beam. Images used to calibrate the number of counts per electron incident on the CCD's phosphor are also insensitive to things beam properties such as astigmatism, beam tilt, etc.

The Faraday cup readings were reported in nanoamperes (nA) and the images were simply converted into the total counts within each image (with no attempt to account for any background signal). Provided the CCD elements are not near saturation, there will be an average number of counts generated per electron that hits the CCD's phosphor. The plot above estimates this value by converting the nA reading of the Faraday cup into electrons/sec and plotting that vs the number of counts per image for a 1 sec exposure. In the case of the EMC's 3200FS, the number of counts per incident electron is 6.74.

This value is significantly different from both the value determined when the CCD camera was installed and from an earlier estimate during the calibration of serialEM on this microscope. However, we reset both these programs to reflect this more accurate calibration. At least for the time immediately after this Faraday cup calibration was performed, the reading of the pico-ampmeter when the focusing screen of the 3200FS is inserted was also adjusted so that when the meter is read at a magnification of 40,000x, the reported pA/cm2 is the same as the electrons/Å2 that the specimen experiences.