Replacement Camera

The image recorded using an exposure time of 1.4 s is not perfect, but the fact that we could get an image that appeared this good gave us hope that we could figure out the problem.

We knew that the black (or white) artifacts in these images were not bad pixels in the CCD since an image with no illumination did not show them. Except for the observation that we could not make the largest of the artifacts vanish with new gain references, these artifacts behave essentially as dirt on the sensor would (and we looked into ways to clean the sensor). We further played with different electron doses and exposure times, and convinced ourselves that the problem was simply the number of electrons that the CCD was trying to record. It finally occurred to us that the black/white contrast reversal in the artifacts was happening right around an electron dose that matched the target of 10,000 counts/pixel set for the gain reference.

It also occurred to us that the gain reference was being recorded at an electron dose that might be too close to the saturation point of the CCD (this 14 bit dynamic range camera should saturate at 16,000, but actual images seem to have fewer than ~14,000 counts/pixel). Indeed, for the UltraScan 4000 on the JEOL JEM 3200FS, a target of 10,000 is < 20% of the level of saturation, while that same target for the 1010's replacement camera is ~70% of saturation.

When we lowered the target for the gain reference, the images looked significantly better: we lost the horrible black artifacts and obtained good images regardless of the electron dose (except when the sensor is saturated). We tried several different gain reference targets, and finally settled on a target value of 7000.

Calibration of Magnifications

Once we had solved the problem with the gain references and the overall appearance of images collected using the replacement camera, we needed to calibrate the different magnifications. Calibrating a camera with DigitalMicrograph is done by measuring known distances with some sort of calibration grid and turning those known distances into pixel sizes and the pixel size into an apparent magnification.

For the lower mags, spacings between the lines in a replica diffraction grating (a "waffle grid") and/or the diameter of latex beads (if any are present) can be used. For higher mags, the atomic spacings in various crystals (catalase, asbestos, Au, etc.) can be used (and these measurements are actually done in Fourier space). For the lower mags, there must be at least one diffraction grating line present in an image used for calibration (and significantly more is much better), and "lower mags" really means "magnifications low enough to have 1 or 2 lines in an image. On the JEOL JEM 3200FS (with 15 μm pixels and a 4k x 4k field of view), one can use images up to ~25,000x for this purpose. For the higher magnifications, the spacings from polycrystalline Au/Pd on the waffle grids becomes visible at 100,000x and higher. However, for this replacement camera, with larger pixels (24 μm) and a 1k x 1k field of view, the highest magnification that can be used with the waffle grid's diffraction grating lines is ~12,000x, and we were unable to see any atomic spacings at high magnifications where we attempted to record calibration images. Because of these constraints, the replacement camera was calibrated using images acquired in normal Mag mode, starting at 600x and ending at 12,000x. We offer a collection of magnifications and pixel sizes from this calibration.

While acquiring images to calibrate the new camera, we noticed that there was a significant difference in magnification for the two magnifications (600x and 1000x) that appear in both the normal Mag mode magnifications and in the Low Mag mode magnifications. For example, the images below are both nominally recorded at 1000x: the image at the left was acquired at the 1000x magnification in the normal Mag mode settings (the third available mag) while the image at the right was recorded at the highest possible Low Mag mode magnification, also nominally 1000x. These images are clearly nowhere near the same magnification, and based on measuring the length of 20 "waffle squares," the image at the left is ~1.27x higher magnification than the image on the right.

An image recorded on the JEOL JEM 1010 at 1000x magnification in Normal Mag mode, much more detailed than with the Low Mag mode.
An image recorded on the JEOL JEM 1010 at 1000x magnification in Low Mag mode, less detailed than with the Normal Mag mode.

We will have a JEOL service engineer who works on the 1010 investigate this issue. At this time, we do not know whether the Low Mag mode magnifications are all slightly off, or if some of the lower normal Mag magnifications are incorrect.