Machine Vision Software Enables Normalization of Electron Dose Calibration Between Microscopes and Delivers Accurate Quantifiable Tracking of Electron Dose for In-Situ, Operando, and Dose Sensitive Experiments

Although transmission electron microscopy (TEM) relies on the interaction of an electron beam with the sample to produce its resulting image, the beam can detrimentally affect the sample through atomic displacement, radiolysis, heat transfer, charging, and contamination [1]. Low-impact (low-dose) imaging techniques, which minimize the number of electrons required to generate an image or series of images, are especially important for fragile samples such as polymers, biological specimens and 2D materials. While most applications, such as cryo-TEM for example, are primarily concerned with the cumulative dose (electrons/Å2), in-situ and operando experiments, particularly liquid-EM, are highly sensitive to the dose rate, or fluence (electrons/Å2s-1) [2]. Efforts to quantify both the cumulative dose and dose rate and their respective effect on experimental results are well documented in the literature [2–5]. Improvements in detector sensitivity, development of stochastic imaging workflows, and post-processing routines have greatly contributed to being able to obtain high resolution TEM images of traditionally beam sensitive sample [6,7]. However, despite these technical advances, a meaningful method of dose calibration across microscope platforms combined with a practical way to quantify the delivered dose over a course of an experiment has, until now, remained a significant impediment in dose management. First, manual calibration a TEM’s beam current is a tedious, multistep process that is difficult to normalize between microscopes due to reliance on screen current measurements and camera detection efficiency as a means of estimating the beam current. Krause et al. reported that integrated screen current measurements are often wildly inaccurate, making them unsuitable for dose calibration [8]. Second, each lens condition (aperture, spot size, magnification etc.) must be individually calibrated and those values must be carefully recorded for each image. In practical terms, this limits the normal operation of the TEM. Thus, some form of assisting technology is necessary both to improve the speed and accuracy of the calibration process and align the resulting dose metadata to each image automatically. AXON Dose is a machine-vision software paired with a dedicated Faraday-cup calibration holder [9]. It is designed to calibrate and index the electron dose conditions throughout the course of a TEM experiment on a per-pixel basis while compensating for sample drift and mechanical slop. Accurate calibration of the beam current is performed with a dedicated calibration holder that contains an integrated Faraday cup in the sample plane and a through-hole for measuring the beam area. Multiple lens conditions can be calibrated during a semi-automated calibration process resulting in comprehensive dose profile for the microscope. The AXON software monitors the imaging conditions and accumulated dose by tracking the beam’s position on the sample, utilizing machine vision to reregister cumulative dose history against sample features. Dose information is saved to each image and can be easily visualized both graphically and through color coded heatmap indicators that can be applied to the image. Fig. 1 shows an example of the user interface, which allows the users to easily visualize the dose exposure at any point in the image series and graphically visualize any of the collected metadata associated with the images. The AXON Dose platform provides a state-of-the-art solution to calibrating, monitoring, and tracking both the electron fluence and the total dose delivered to specific areas of the sample on a pixel-by-pixel basis. This ability unlocks a new paradigm for imaging dose sensitive samples and elucidating influence of the electron beam on a sample. AXON Dose is a comprehensive dose calibration and management software. (A) A dedicated calibration holder is used to calibrate lens conditions via a semi-automated program. The tip of the sample holder moves back and forth between and integrated Faraday-cup for current measurement and a through hole for beam area measurement. B. Electron dose exposure is easily visualized in any image with color-coded heat map overlays. Cumulative and incident dose can be plotted graphically on an experimental timeline enabling the user to holistically identify trends and behaviors that occur over the TEM imaging session.