MASDET2‐ software for quantitativeSTEMimaging
Authors: Krzyzanek Vladislav , Tacke Sebastian 
Published: December 2016

The idea of mass determination using a TEM was first proposed by E. Zeitler and G. F. Bahr [1]. The principle based on the single electron scattering approach (approximately linear relationship between the fraction of incident electrons scattered by a thin proteinaceous specimen and its molecular mass) was realized experimentally later by dedicated scanning transmission electron microscopes (STEM) equipped by a field emission gun and an annular dark‐field (ADF) detector (for actual review see [2]). Besides the aforementioned technical realization, several mass determination software packages for the data analysis were developed independently – IMPSYS [3] and PCMass [4]. This idea for mass determination was later applied to so‐called low voltage STEM by extending a commercial SEM by a proper ADF detector [5] and dedicated software MASDET [6] that worked on the linear approximation like IMPSYS. Here, we report on extensions of MASDET program that uses nonlinear relations and significantly extends applicable range of thickness.Typically, programs using the single electron scattering approach give only exact results for samples not thicker than a few percent of the mean‐free‐path‐length (MFPL) of the impinging electrons. One improvement was introduced in MASDET, using a so‐called linearization, where the applicable range of thickness was extended to approximately 2x MFPL [6]. Here, we present MASDET version 2 which avoids the limitations of the linear approximation by determining the nonlinear relation between signal and mass with the help of Monte Carlo simulation software packages like MONCA [7]. With this improvement we may reach thicknesses approximately 7x MFPL (note that this depends on the detection geometry of the STEM detector).As the previous version, MASDET2 has a user‐friendly graphical interface allowing almost all operations to be done by mouse. It relies on MATLAB (MathWorks), and therefore should run under all systems suitable for MATLAB. MASDET2 allows the mass of specified regions of interest (ROI) to be calculated conveniently and in a highly automated manner from the digital ADF micrographs and recording parameters. Three procedures are available allowing the calculation of the mass‐per‐area (MPA) of sheet‐like structures, the mass‐per‐length (MPL) of filaments, and the mass‐per‐particle (MPP) of globular particles.Note that all calculations are done as absolute measurements without any mass calibration standards. Figure 1 shows the proof‐of‐principle measurement. Here, Tobacco Mosaic Virus (TMV) was investigated. For the mass calculations the relation of the ADF signal vs thickness was simulated using MONCA (for comparison a relation for lipid is shown on Figure 1b; the used SEM is S‐5000 (Hitachi) equipped by a home‐made ADF detector, operating at 30 keV). With this relation, the recorded signal can be converted into a mass value which can be finally added up in each ROI. Figure 1c shows the final result of the MPL determination which is 126±4 kDa/nm. After correction of the beam‐induced mass loss, see figure 1d, which can also be done within the new version of MASDET2 software, the MPL value of TMV was corrected to 131±6 kDa/nm which represents the theoretical MPL value of TMV, see also [8].